Il-1 family variants

ABSTRACT

The present invention provides compositions and methods relating to IL-1Rrp2 requiring proteins.

This application claims the benefit of U.S. provisional patentapplication No. 60/843,311, filed Sep. 8, 2006 which is herebyincorporated by reference.

FIELD OF THE INVENTION

This application provides nucleic acids, polypeptides, compositions,assays, and methods relating to variants of IL-1 Family members thatsignal through IL-1Rrp2.

BACKGROUND OF THE INVENTION

The IL-1 family includes several cytokines whose primary function is tomediate immune and inflammatory responses. The earliest membersdiscovered were IL-1 alpha, IL-1 beta, IL-1 receptor antagonist(IL-1ra), and IL-18 (previously known as IGIF and sometimes IL-1 gamma).Following the discovery of additional proteins with homology to theseIL-1 family members, a nomenclature system was adopted in which IL-1alpha is referred to as IL-1F1, IL-1 beta as IL-1F2, IL-1ra as IL-1F3and IL-18 as IL-1F4. Seven additional cytokines have been classified asIL-1 family members based on amino acid sequence similarity, identity ofgene structure, and predicted or known three-dimensional structure(Sims, J. E. et al., Trends Immunol 22:537, 2001; Dunn, E., et al.,Trends Immunol 22:533, 2001; Dunn, E. F., et al., Biochemistry 42:10938,2003; Schmitz et al. Immunity 23:479-490, 2005).

IL-1 alpha, IL-1 beta and IL-1ra (IL-1F1-3, respectively) bind toreceptors that are members of the immunoglobulin superfamily, the 80 kDatype I receptor (IL-1RI) and a 68 kDa type II receptor (IL-1RII), aswell as a soluble proteolytic fragment of IL-1RII (sIL-1RII). Binding ofIL-1 (alpha or beta) to the type I IL-1 receptor (IL-1R) results inrecruitment of the IL-1R homolog, IL-1R accessory protein (IL-1RAcP orAcP), which does not directly bind the ligands but is required forsignal transduction (Sims et al. Trends Immunol 22; 537, 2001); bindingof IL-1ra does not. Signaling by IL-18 is very similar, although IL-18utilizes a different receptor complex (Born, T. L., et al., J Biol Chem273:29445, 1998). IL-1F5, F6, F8 and F9 make use of the IL-1R-relatedprotein 2 (IL-1Rrp2), with F6, F8 and F9 agonizing this receptorpathway, and IL-1F5 antagonizing it (Debets, R., et al., J Immunol167:1440, 2001; Towne et al. 2004 J Biol Chem 279(14):13677)

Several members of the IL-1 family (IL-1 alpha, IL-1 beta, IL-18, IL-1F7and IL-33) are synthesized as precursor molecules that areproteolytically cleaved, by caspase-1 in the case of IL-1 beta andIL-18, and by an unidentified protease or proteases for IL-33, IL-1alpha and IL-1F7. IL-1ra is activated by signal peptidase cleavage of ashort peptide from the n-terminus. However, little is known about what,if any, processing occurs with the remaining family members.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an alignment of the N-terminal portions of wild typeIL-1F5, F6, F8 and F9. There is a (Met or Ile)-Xaa-Asp sequence presentin each of F5, F6, F8 and F9, marked by underlining of the Met/Ile andthe Asp residues. There is a similar “aliphatic amino acid-X-Aspartateor other polar amino acid” motif present in all IL-1 family members, andthis can be used to align IL-1 family sequences. The consensus motif isindicated by @XD where @ may be an aliphatic amino acid such as Met orIle and X is any one amino acid D is Asp. In FIG. 1, the sequences arelined up using the Met/Ile-Xaa-Asp motif (Met 11 in F5, Ile15 in F6,Ile14 in F8, and Ile27 in F9 respectively), so that the naturalN-termini (with initiating methionines) lie at different distancesupstream of the aliphatic amino acid, @, of the motif

FIG. 2 is the full length wild type amino acid sequences of IL-1F5,IL-1F6, IL-1F8 and IL-1F9.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated IL-1F5polypeptide that antagonizes signal transduction/activation throughIL-1Rrp2, where the IL-1F5 polypeptide contains the sequenceMet-Lys-Asp, which matches the consensus @XD depicted in FIG. 1, andwherein the polypeptide comprises nine amino acids on the N-terminalside of the above-referenced methionine. In one embodiment the IL-1F5polypeptide is a human IL-1F5 polypeptide. In one embodiment, the IL-1F5polypeptide of the invention comprises an amino acid sequence having amethionine at position ten of the amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one of the aminoacid sequence. In one embodiment, the IL-1F5 polypeptide of theinvention comprises an amino acid sequence having a methionine atposition ten of the amino acid sequence, position ten being relative tothe N-terminal amino acid at position one of the amino acid sequence andan amino acid selected from the group consisting of valine andmethionine at the N-terminal amino acid at position one. In oneembodiment, the IL-1F5 polypeptide of the invention comprises an aminoacid sequence having a methionine at position ten of the amino acidsequence, position ten being relative to the N-terminal amino acid atposition one of the amino acid sequence and a leucine at position two ofits amino acid sequence. In a particular embodiment the IL-1F5polypeptide of the invention, comprises an amino acid sequence having amethionine at position ten of the amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one of the aminoacid sequence and an amino acid selected from the group consisting ofvaline and methionine at the N-terminal amino acid at position and aleucine at position two.

In particular embodiments, the IL-1F5 polypeptide of the inventioncomprises at least 90%, at least 95%, at least 98% or at least 99%identity to SEQ ID NO 1 and a methionine at position ten of the aminoacid sequence of the IL-1F5 polypeptide of the invention, position tenbeing relative to the N-terminal amino acid a position one. In someembodiments, the isolated IL-1F5 polypeptide of the inventionantagonizes signal transduction/activation through IL-1Rrp2 more thanthe IL-1F5 polypeptide having the amino acid sequence of SEQ ID NO 1. Insome embodiments, the isolated IL-1F5 polypeptide of the inventionantagonizes signal transduction/activation through IL-1Rrp2 more thanabout 5 fold, 10 fold, 100 fold, 1,000 fold the level of antagonizationof signal transduction/activation of the IL-1F5 polypeptide having theamino acid sequence of SEQ ID NO 1. The level of signaltransduction/activation antagonization is measured according to themethod described in Example 2A.

In a particular embodiment, the isolated IL-1F5 polypeptide of theinvention has a methionine at position ten of its amino acid sequencerelative to the N-terminal amino acid at position one and comprises anamino acid sequence selected from the group consisting of:

(SEQ ID NO 6) VLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCLSCGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQLPENGGWNAPITDFYF QQCD, (SEQ ID NO 7)MLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCLSCGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQLPENGGWNAPITDFYF QQCDDYKDDDDKHHH,(SEQ ID NO 8) MLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCLSCGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQLPENGGWNAPITDFYF QQCD  and(SEQ ID NO 9) MLSGALCFRMKDSALKVLYLHNNQLLAGGLHAGKVIKGEEISVVPNRWLDASLSPVILGVQGGSQCLSCGVGQEPTLTLEPVNIMELYLGAKESKSFTFYRRDMGLTSSFESAAYPGWFLCTVPEADQPVRLTQLPENGGWNAPITDFYF QQCDDYKDDDDKHHH.

In another embodiment, a nucleic acid sequence encoding the isolatedIL-1F5 polypeptide of the invention is provided. In another aspect ofthe invention, a recombinant vector that directs expression of a nucleicacid encoding an isolated IL-1F5 polypeptide of the invention isprovided. In a particular embodiment, the vector of the inventioncomprises a nucleic acid sequence selected from the group consisting of:

(SEQ ID NO 69) GTCCTGAGTGGGGCGCTGTGCTTCCGAATGAAGGACTCGGCATTGAAGGTGCTTTATCTGCATAATAACCAGCTTCTAGCTGGAGGGCTGCATGCAGGGAAGGTCATTAAAGGTGAAGAGATCAGCGTGGTCCCCAATCGGTGGCTGGATGCCAGCCTGTCCCCCGTCATCCTGGGTGTCCAGGGTGGAAGCCAGTGCCTGTCATGTGGGGTGGGGCAGGAGCCGACTCTAACACTAGAGCCAGTGAACATCATGGAGCTCTATCTTGGTGCCAAGGAATCCAAGAGCTTCACCTTCTACCGGCGGGACATGGGGCTCACCTCCAGCTTCGAGTCGGCTGCCTACCCGGGCTGGTTCCTGTGCACGGTGCCTGAAGCCGATCAGCCTGTCAGACTCACCCAGCTTCCCGAGAATGGTGGCTGGAATGCCCCCATCACAGACTTCTACTTCCAGCAGTGTGACTAA  and (SEQ ID NO 70)ATGCTGAGTGGGGCGCTGTGCTTCCGAATGAAGGACTCGGCATTGAAGGTGCTTTATCTGCATAATAACCAGCTTCTAGCTGGAGGGCTGCATGCAGGGAAGGTCATTAAAGGTGAAGAGATCAGCGTGGTCCCCAATCGGTGGCTGGATGCCAGCCTGTCCCCCGTCATCCTGGGTGTCCAGGGTGGAAGCCAGTGCCTGTCATGTGGGGTGGGGCAGGAGCCGACTCTAACACTAGAGCCAGTGAACATCATGGAGCTCTATCTTGGTGCCAAGGAATCCAAGAGCTTCACCTTCTACCGGCGGGACATGGGGCTCACCTCCAGCTTCGAGTCGGCTGCCTACCCGGGCTGGTTCCTGTGCACGGTGCCTGAAGCCGATCAGCCTGTCAGACTCACCCAGCTTCCCGAGAATGGTGGCTGGAATGCCCCCATCACAGACTTCTACTTCCAGCAGTGTGACAGATCTGGCAGTTCTGACTACAAGGACGACGACGACAAGGGCAGTTCTCACCATCACCATCACCACTAG.In another aspect, a host cell transfected or transduced with arecombinant vector that directs expression of a nucleic acid encoding anIL-1F5 polypeptide of the invention is provided. In another aspect, amethod of producing an isolated IL-1F5 polypeptide of the inventioncomprising culturing the host cell transfected or transduced with arecombinant vector that directs expression of an IL-1F5 polypeptide ofthe invention, under conditions promoting expression and isolating theexpressed IL-1F5 polypeptide is provided.

An antibody or a fragment thereof that specifically binds an IL-1F5polypeptide and prevents IL-1F5 antagonism of signal transductionthrough IL-1Rrp2 is provided. In particular embodiments, an antibodythat binds an IL-1F5 polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO 6, SEQ ID NO 7, SEQ IDNO 8, and SEQ ID NO 9 is provided. In some embodiments, the IL-1F5antibody of the invention, is a monoclonal antibody, particularly achimeric antibody, a humanized antibody or a fully human antibody. Theinvention provides a composition, particularly a pharmaceuticalcomposition, comprising the IL-1F5 antibody of the invention and aphysiologically acceptable diluent, excipient or carrier. A method ofstimulating the immune system of an immunosuppresed subject, comprisingadministering the anti-IL-1F5 antibody of the invention to animmunosuppresed subject in an amount sufficient to stimulate thepatient's immune system is provided.

In another aspect, a composition, particularly a pharmaceuticalcomposition, comprising an IL-1F5 polypeptide of the invention and aphysiologically acceptable diluent, excipient or carrier is provided. Inanother embodiment, the invention provides a method of treating aninflammatory or autoimmune condition in a subject wherein theinflammatory or autoimmune condition is mediated by IL-1Rrp2, comprisingadministering to the subjecting an amount of the IL-1F5 polypeptide ofthe invention sufficient to reduce at least one symptom of theinflammatory or autoimmune condition in the subject. In one embodiment,the condition to be treated is an inflammatory condition of the skin,lungs or airways mediated by IL-1Rrp2. In a particular embodiment, thecondition to be treated is selected from the group consisting ofpsoriasis, seborrheic dermatitis, atopic dermatitis, including chronicatopic dermatitis, allergic contact dermatitis, lichen simplexchronicus, pityriasis rubra pilaris, nummular dermatitis, asthma,allergic rhinitis, gastro-esophageal reflux disease, arthriticconditions including, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis. In particular embodiments of the above methods, thesubject is human.

In one aspect, the present invention provides an isolated IL-1F6polypeptide that agonizes signal transduction/activation throughIL-1Rrp2, where the IL-1F6 polypeptide contains the sequenceIle-Gln-Asp, which matches the consensus @XD described in FIG. 1, andwherein the polypeptide comprises nine amino acids on the N-terminalside of the above-referenced isoleucine. In one embodiment the IL-1F6polypeptide is a human IL-1F6 polypeptide. In one embodiment, the IL-1F6polypeptide of the invention comprises an amino acid sequence having anisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one. In oneembodiment, the IL-1F6 polypeptide of the invention comprises an aminoacid sequence having an isoleucine at position ten of its amino acidsequence, position ten being relative to the N-terminal amino acid atposition one and an amino acid selected from the group consisting oflysine and methionine at the N-terminal amino acid at position one. Inone embodiment, the IL-1F6 polypeptide of the invention comprises anamino acid sequence having an isoleucine at position ten of its aminoacid sequence, position ten being relative to the N-terminal amino acidat position one, and an isoleucine at position two of its amino acidsequence. In a particular embodiment the IL-1F6 polypeptide of theinvention, comprises an amino acid sequence having a isoleucine atposition ten of the amino acid sequence, position ten being relative tothe N-terminal amino acid at position one, and an amino acid selectedfrom the group consisting of lysine and methionine at the N-terminalamino acid at position and an isoleucine at position two.

In particular embodiments, the isolated IL-1F6 polypeptide comprises atleast 90%, at least 95%, at least 97% or at least 98% identity to SEQ IDNO 2 or to SEQ ID NO 2 except that there is an arginine at amino acidposition 12 rather than a glutamine and an isoleucine at position ten,position ten being relative to the N-terminal amino acid at position oneof the IL-1F6 amino acid sequence of the invention. In some embodiments,the isolated IL-1F6 polypeptide of the invention agonizes signaltransduction/activation through IL-1Rrp2 more than the IL-1F6polypeptide having the N-terminal amino acid sequence of SEQ ID NO 2. Insome embodiments, the isolated IL-1F6 polypeptide of the inventionagonizes signal transduction/activation through IL-1Rrp2 more than about5 fold, 10 fold, 100 fold, 200 fold, 1,000 fold, 2,000 fold, 10,000fold, 50,000 fold the level of agonization of signaltransduction/activation of the IL-1F6 polypeptide having the amino acidsequence of SEQ ID NO 2. The level of signal transduction/activationagonization is measured according to the method described in Example 2B.

In another aspect, the invention provides an isolated IL-1F6 polypeptidethat agonizes signal transduction/activation through IL-1Rrp2, whereinthe IL-1F6 polypeptide comprises an amino acid sequence having aisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one and an aminoacid sequence selected from the group consisting of:

(SEQ ID NO 10) KIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLF, (SEQ ID NO 11)KIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLFDYKDDDDKHHH,(SEQ ID NO 12) MIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLF, (SEQ ID NO 13)MIDTPQQGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLFDYKDDDDKHHH,(SEQ ID NO 65) KIDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLF, (SEQ ID NO 66)KIDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLFDYKDDDDKHHH,(SEQ ID NO 67) MIDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLF  and(SEQ ID NO 68) MIDTPQRGSIQDINHRVWVLQDQTLIAVPRKDRMSPVTIALISCRHVETLEKDRGNPIYLGLNGLNLCLMCAKVGDQPTLQLKEKDIMDLYNQPEPVKSFLFYHSQSGRNSTFESVAFPGWFIAVSSEGGCPLILTQELGKANTTDFGLT MLFDYKDDDDKHHH.

In another embodiment, a nucleic acid sequence encoding the isolatedIL-1F6 polypeptide of the invention is provided. In another aspect ofthe invention, a recombinant vector that directs expression of a nucleicacid encoding an isolated IL-1F6 polypeptide of the invention isprovided. In a particular embodiment, the vector of the inventioncomprises a nucleic acid sequence selected from the group consisting of:

(SEQ ID NO 71) AAAATTGACACACCTCAGCGGGGGAGCATTCAGGATATCAATCATCGGGTGTGGGTTCTTCAGGACCAGACGCTCATAGCAGTCCCGAGGAAGGACCGTATGTCTCCAGTCACTATTGCCTTAATCTCATGCCGACATGTGGAGACCCTTGAGAAAGACAGAGGGAACCCCATCTACCTGGGCCTGAATGGACTCAATCTCTGCCTGATGTGTGCTAAAGTCGGGGACCAGCCCACACTGCAGCTGAAGGAAAAGGATATAATGGATTTGTACAACCAACCCGAGCCTGTGAAGTCCTTTCTCTTCTACCACAGCCAGAGTGGCAGGAACTCCACCTTCGAGTCTGTGGCTTTCCCTGGCTGGTTCATCGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTACCCAAGAACTGGGGAAAGCCAACACTACTGACTTTGGGTTAACT ATGCTGTTTTAA  and(SEQ ID NO 72) ATGATTGACACACCTCAGCGGGGGAGCATTCAGGATATCAATCATCGGGTGTGGGTTCTTCAGGACCAGACGCTCATAGCAGTCCCGAGGAAGGACCGTATGTCTCCAGTCACTATTGCCTTAATCTCATGCCGACATGTGGAGACCCTTGAGAAAGACAGAGGGAACCCCATCTACCTGGGCCTGAATGGACTCAATCTCTGCCTGATGTGTGCTAAAGTCGGGGACCAGCCCACACTGCAGCTGAAGGAAAAGGATATAATGGATTTGTACAACCAACCCGAGCCTGTGAAGTCCTTTCTCTTCTACCACAGCCAGAGTGGCAGGAACTCCACCTTCGAGTCTGTGGCTTTCCCTGGCTGGTTCATCGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTACCCAAGAACTGGGGAAAGCCAACACTACTGACTTTGGGTTAACTATGCTGTTTAGATCTGGCAGTTCTGACTACAAGGACGACGACGACAAGGGCAGTTCTCACCATCACCATCACCACTAG.In another aspect, a host cell transfected or transduced with arecombinant vector that directs expression of a nucleic acid encoding anIL-1F6 polypeptide of the invention is provided. In anther aspect, amethod of producing an isolated IL-1F6 polypeptide of the inventioncomprising culturing the host cell transfected or transduced with arecombinant vector that directs expression of an IL-1F6 polypeptide ofthe invention, under conditions promoting expression and isolating theexpressed IL-1F6 polypeptide is provided.

In another aspect, a composition, particularly a pharmaceuticalcomposition, comprising an IL-1F6 polypeptide of the invention and aphysiologically acceptable diluent, excipient or carrier is provided. Amethod of stimulating the immune system of an immunosuppresed subject,comprising administering the IL-1F6 polypeptide according of theinvention to an immunosuppresed subject in an amount sufficient tostimulate the subject's immune system is provided.

An antibody that specifically binds IL-1F6, where IL-1F6 may be a fulllength IL-1F6 or a truncant of full length IL-1F6, and preventsproteolytic cleavage thereof, particularly proteolytic cleavage to amore active form where activity is relative to that of the full lengthIL-1F6, is provided. An antibody that binds an IL-1F6 polypeptide of theinvention, and prevents signal transduction/activation through IL-1FRrp2is also provided. In some embodiments, an antibody binding an IL-1F6polypeptide selected from the group consisting of: SEQ ID NO 10, SEQ IDNO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO67 and SEQ ID NO 68 is provided. In some embodiments, the IL-1F6antibody of the invention, is a monoclonal antibody, particularly achimeric antibody, a humanized antibody or a fully human antibody. Theinvention provides a composition, particularly a pharmaceuticalcomposition, comprising the IL-1F6 antibody of the invention and aphysiologically acceptable diluent, excipient or carrier.

The invention also provides, a method of treating an inflammatory orautoimmune condition mediated by IL-1Rrp2, comprising administering theIL-1F6 antibody of the invention to a subject in an amount sufficient toameliorate at least one symptom mediated by IL-1Rrp2 of the condition.In one embodiment, the condition to be treated is an inflammatorycondition of the skin, lungs or airways mediated by IL-1Rrp2. In aparticular embodiment, the condition to be treated is selected from thegroup consisting of psoriasis, seborrheic dermatitis, atopic dermatitis,including chronic atopic dermatitis, allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris, nummular dermatitis,asthma, allergic rhinitis, gastro-esophageal reflux disease, arthriticconditions including, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis. In particular embodiments, the subject to be treated inthe above methods is a human.

In one aspect, the present invention provides an isolated IL-1F8polypeptide that agonizes signal transduction/activation throughIL-1Rrp2, where the IL-1F8 polypeptide contains the sequenceIle-Arg-Asp, which matches the consensus @XD described in FIG. 1, andwherein the polypeptide comprises nine amino acids on the N-terminalside of the above-referenced isoleucine. In one embodiment the IL-1F8polypeptide is a human IL-1F8 polypeptide. In one embodiment, the IL-1F8polypeptide of the invention comprises an amino acid sequence having anisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one. In oneembodiment, the IL-1F8 polypeptide of the invention comprises an aminoacid sequence having an isoleucine at position ten of its amino acidsequence, position ten being relative to the N-terminal amino acid atposition one and an amino acid selected from the group consisting ofarginine and methionine at the N-terminal amino acid at position one. Inone embodiment, the IL-1F8 polypeptide of the invention comprises anamino acid sequence having an isoleucine at position ten of its aminoacid sequence, position ten being relative to the N-terminal amino acidat position one, and a glutamic acid at position two of its amino acidsequence. In a particular embodiment the IL-1F8 polypeptide of theinvention, comprises an amino acid sequence having a isoleucine atposition ten of the amino acid sequence, position ten being relative tothe N-terminal amino acid at position one, and an amino acid selectedfrom the group consisting of arginine and methionine at the N-terminalamino acid at position and an glutamic acid at position two.

In particular embodiments, the isolated IL-1F8 polypeptide comprises atleast 90%, at least 95%, or at least 98% identity to SEQ ID NO 3 and anisoleucine at position ten, position ten being relative to theN-terminal amino acid at position one of the IL-1F8 amino acid sequenceof the invention. In some embodiments, the isolated IL-1F8 polypeptideof the invention agonizes signal transduction/activation throughIL-1Rrp2 more than the IL-1F8 polypeptide having the amino acid sequenceof SEQ ID NO 3. In some embodiments, the isolated IL-1F8 polypeptide ofthe invention agonizes signal transduction/activation through IL-1Rrp2more than about 5 fold, 10 fold, 100 fold, 200 fold, 1,000 fold, 3,000fold, 5,000 fold, 10,000 fold, 50,000 fold the level of agonization ofsignal transduction/activation of the IL-1F8 polypeptide having theamino acid sequence of SEQ ID NO 3. The level of signaltransduction/activation agonization is measured according to the methoddescribed in Example 2B.

In another aspect, the invention provides an isolated IL-1F8 polypeptidethat agonizes signal transduction/activation through IL-1Rrp2, whereinthe IL-1F8 polypeptide comprises an amino acid sequence having aisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one and an aminoacid sequence selected from the group consisting of:

(SEQ ID NO 14) REAAPKSYAIRDSRQMVWVLSGNSLIAAPLSRSIKPVTLHLIACRDTEFSDKEKGNMVYLGIKGKDLCLFCAEIQGKPTLQLKEKNIMDLYVEKICAQKPFLFFHNKEGSTSVFQSVSYPGWFIATSTTSGQPIFLTKERGITNNTNFYL DSVE, (SEQ ID NO 15)REAAPKSYAIRDSRQMVWVLSGNSLIAAPLSRSIKPVTLHLIACRDTEFSDKEKGNMVYLGIKGKDLCLFCAEIQGKPTLQLKEKNIMDLYVEKKAQKPFLFFHNKEGSTSVFQSVSYPGWFIATSTTSGQPIFLTKERGITNNTNFYLD SVEDYKDDDDKHHH;(SEQ ID NO 16) MEAAPKSYAIRDSRQMVWVLSGNSLIAAPLSRSIKPVTLHLIACRDTEFSDKEKGNMVYLGIKGKDLCLFCAEIQGKPTLQLKEKNIMDLYVEKKAQKPFLFFHNKEGSTSVFQSVSYPGWFIATSTTSGQPIFLTKERGITNNTNFYLD SVE  and(SEQ ID NO 17) MEAAPKSYAIRDSRQMVWVLSGNSLIAAPLSRSIKPVTLHLIACRDTEFSDKEKGNMVYLGIKGKDLCLFCAEIQGKPTLQLKEKNIMDLYVEKKAQKPFLFFHNKEGSTSVFQSVSYPGWFIATSTTSGQPIFLTKERGITNNTNFYLD SVEDYKDDDDICHHH.

In another embodiment, a nucleic acid sequence encoding the isolatedIL-1F8 polypeptide of the invention is provided. In another aspect ofthe invention, a recombinant vector that directs expression of a nucleicacid encoding an isolated IL-1F8 polypeptide of the invention isprovided. In a particular embodiment, the vector of the inventioncomprises a nucleic acid sequence selected from the group consisting of:

(SEQ ID NO 73) CGCGAGGCAGCACCCAAATCCTATGCTATTCGTGATTCTCGACAGATGGTGTGGGTCCTGAGTGGAAATTCTTTAATAGCAGCTCCTCTTAGCCGCAGCATTAAGCCTGTCACTCTTCATTTAATAGCCTGTAGAGACACAGAATTCAGTGACAAGGAAAAGGGTAATATGGTTTACCTGGGAATCAAGGGAAAAGATCTCTGTCTCTTCTGTGCAGAAATTCAGGGCAAGCCTACTTTGCAGCTTAAGGAAAAAAATATCATGGACCTGTATGTGGAGAAGAAAGCACAGAAGCCCTTTCTCTTTTTCCACAATAAAGAAGGCTCCACTTCTGTCTTTCAGTCAGTCTCTTACCCTGGCTGGTTCATAGCCACCTCCACCACATCAGGACAGCCCATCTTTCTCACCAAGGAGAGAGGCATAACTAATAACACTAACTTCTACTTAGATTCTGTGGAATAA  and (SEQ ID NO 74)ATGGAGGCAGCACCCAAATCCTATGCTATTCGTGATTCTCGACAGATGGTGTGGGTCCTGAGTGGAAATTCTTTAATAGCAGCTCCTCTTAGCCGCAGCATTAAGCCTGTCACTCTTCATTTAATAGCCTGTAGAGACACAGAATTCAGTGACAAGGAAAAGGGTAATATGGTTTACCTGGGAATCAAGGGAAAAGATCTCTGTCTCTTCTGTGCAGAAATTCAGGGCAAGCCTACTTTGCAGCTTAAGGAAAAAAATATCATGGACCTGTATGTGGAGAAGAAAGCACAGAAGCCCTTTCTCTTTTTCCACAATAAAGAAGGCTCCACTTCTGTCTTTCAGTCAGTCTCTTACCCTGGCTGGTTCATAGCCACCTCCACCACATCAGGACAGCCCATCTTTCTCACCAAGGAGAGAGGCATAACTAATAACACTAACTTCTACTTAGATTCTGTGGAAGGATCTGGCAGTTCTGACTACAAGGACGACGACGACAAGGGCAGTTCTCACCATCACCATCACCACTAG.In another aspect, a host cell transfected or transduced with arecombinant vector that directs expression of a nucleic acid encoding anIL-1F8 polypeptide of the invention is provided. In another aspect, amethod of producing an isolated IL-1F8 polypeptide of the inventioncomprising culturing the host cell transfected or transduced with arecombinant vector that directs expression of an IL-1F8 polypeptide ofthe invention, under conditions promoting expression and isolating theexpressed IL-1F8 polypeptide is provided.

In another aspect, a composition, particularly a pharmaceuticalcomposition, comprising an IL-1F8 polypeptide of the invention and aphysiologically acceptable diluent, excipient or carrier is provided. Amethod of stimulating the immune system of an immunosuppresed subject,comprising administering the IL-1F8 polypeptide according of theinvention to an immunosuppresed subject in an amount sufficient tostimulate the subject's immune system is provided. In particularembodiments of the above methods, the subject is a human.

An antibody that specifically binds IL-1F8, where IL-1F8 may be a fulllength IL-1F8 or a truncant of full length IL-1F8, and preventsproteolytic cleavage thereof, particularly proteolytic cleavage to amore active form where activity is relative to that of the full lengthIL-1F8, is provided. An antibody that binds an IL-1F8 polypeptide of theinvention, and prevents signal transduction/activation through IL-1FRrp2is also provided. In some embodiments, an antibody binding an IL-1F8polypeptide selected from the group consisting of: SEQ ID NO 14, SEQ IDNO 15, SEQ ID NO 16 and SEQ ID NO 17, is provided. In some embodiments,the IL-1F8 antibody of the invention, is a monoclonal antibody,particularly a chimeric antibody, a humanized antibody or a fully humanantibody. The invention provides a composition, particularly apharmaceutical composition, comprising the IL-1F8 antibody of theinvention and a physiologically acceptable diluent, excipient orcarrier.

The invention also provides, a method of treating an inflammatory orautoimmune condition mediated by IL-1Rrp2, comprising administering theIL-1F8 antibody of the invention to a subject in an amount sufficient toameliorate at least one symptom mediated by IL-1Rrp2 of the condition.In one embodiment, the condition to be treated is an inflammatorycondition of the skin, lungs or airways mediated by IL-1Rrp2. In aparticular embodiment, the condition to be treated is selected from thegroup consisting of psoriasis, seborrheic dermatitis, atopic dermatitis,including chronic atopic dermatitis, allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris, nummular dermatitis,asthma, allergic rhinitis, gastro-esophageal reflux disease, arthriticconditions including, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis.

In one aspect, the present invention provides an isolated IL-1F9polypeptide that agonizes signal transduction/activation throughIL-1Rrp2, where the IL-1F9 polypeptide comprises the consensus sequence@XD described in FIG. 1, where the IL-1F9 polypeptide contains thesequence Ile-Asn-Asp, which matches the consensus @XD, and wherein thepolypeptide comprises nine amino acids on the N-terminal side of theabove-referenced isoleucine. In one embodiment the IL-1F9 polypeptide isa human IL-1F9 polypeptide. In one embodiment, the IL-1F9 polypeptide ofthe invention comprises an amino acid sequence having an isoleucine atposition ten of its amino acid sequence, position ten being relative tothe N-terminal amino acid at position one. In one embodiment, the IL-1F9polypeptide of the invention comprises an amino acid sequence having anisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one and an aminoacid selected from the group consisting of serine and methionine at theN-terminal amino acid at position one. In one embodiment, the IL-1F9polypeptide of the invention comprises an amino acid sequence having anisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one, and amethionine at position two of its amino acid sequence. In a particularembodiment the IL-1F9 polypeptide of the invention, comprises an aminoacid sequence having a isoleucine at position ten of the amino acidsequence, position ten being relative to the N-terminal amino acid atposition one, and an amino acid selected from the group consisting ofserine and methionine at the N-terminal amino acid at position and anglutamic acid at position two.

In particular embodiments, the isolated IL-1F9 polypeptide comprises atleast 85% or at least 89% identity to SEQ ID NO 4 and an isoleucine atposition ten, position ten being relative to the N-terminal amino acidat position one of the IL-1F9 amino acid sequence of the invention. Insome embodiments, the isolated IL-1F9 polypeptide of the inventionagonizes signal transduction/activation through IL-1Rrp2 more than theIL-1F9 polypeptide having the amino acid sequence of SEQ ID NO 4. Insome embodiments, the isolated IL-1F9 polypeptide of the inventionagonizes signal transduction/activation through IL-1Rrp2 more than about5 fold, 10 fold, 50 fold, 100 fold, 600 fold, 1,000 fold, 3,000 fold,5,000 fold, 10,000 fold or 50,000 fold the level of agonization ofsignal transduction/activation of the IL-1F9 polypeptide having theamino acid sequence of SEQ ID NO 4. The level of signaltransduction/activation agonization is measured according to the methoddescribed in Example 2B.

In another aspect, the invention provides an isolated IL-1F9 polypeptidethat agonizes signal transduction/activation through IL-1Rrp2, whereinthe IL-1F9 polypeptide comprises an amino acid sequence having aisoleucine at position ten of its amino acid sequence, position tenbeing relative to the N-terminal amino acid at position one and an aminoacid sequence selected from the group consisting of:

(SEQ ID NO 18) SMCKPITGTINDLNQQVWTLQGQNLVAVPRSDSVTPVTVAVITCKYPEALEQGRGDPIYLGIQNPEMCLYCEKVGEQPTLQLKEQKIMDLYGQPEPVKPFLFYRAKTGRTSTLESVAFPDWFIASSKRDQPIILTSELGKSYNTAFELNI ND, (SEQ ID NO 19)SMCKPITGTINDLNQQVWTLQGQNLVAVPRSDSVTPVTVAVITCKYPEALEQGRGDPIYLGIQNPEMCLYCEKVGEQPTLQLKEQKIMDLYGQPEPVKPFLFYRAKTGRTSTLESVAFPPDWFIASSKRDQPIILTSELGKSYNTAFELN INDDYKDDDDKHHH,(SEQ ID NO 20) MMCKPITGTINDLNQQVWTLQGQNLVAVPRSDSVTPVTVAVITCKYPEALEQGRGDPIYLGIQNPEMCLYCEKVGEQPTLQLKEQKIMDLYGQPEPVKPFLFYRAKTGRTSTLESVAFPDWFIASSKRDQPIILTSELGKSYNTAFELNI  ND and(SEQ ID NO 21) MMCKPITGTINDLNQQVWTLQGQNLVAVPRSDSVTPVTVAVITCKYPEALEQGRGDPIYLGIQNPEMCLYCEKVGEQPTLQLKEQKIMDLYGQPEPVKPFLFYRAKTGRTSTLESVAFPDWFIASSKRDQPIILTSELGKSYNTAFELNI NDDYKDDDDKHHH.

In another embodiment, a nucleic acid sequence encoding the isolatedIL-1F9 polypeptide of the invention is provided. In another aspect ofthe invention, a recombinant vector that directs expression of a nucleicacid encoding an isolated IL-1F9 polypeptide of the invention isprovided. In a particular embodiment, the vector comprises a nucleicacid sequence selected from the group consisting of:

(SEQ ID NO 75) TCAATGTGTAAACCTATTACTGGGACTATTAATGATTTGAATCAGCAAGTGTGGACCCTTCAGGGTCAGAACCTTGTGGCAGTTCCACGAAGTGACAGTGTGACCCCAGTCACTGTTGCTGTTATCACATGCAAGTATCCAGAGGCTCTTGAGCAAGGCAGAGGGGATCCCATTTATTTGGGAATCCAGAATCCAGAAATGTGTTTGTATTGTGAGAAGGTTGGAGAACAGCCCACATTGCAGCTAAAAGAGCAGAAGATCATGGATCTGTATGGCCAACCCGAGCCCGTGAAACCCTTCCTTTTCTACCGTGCCAAGACTGGTAGGACCTCCACCCTTGAGTCTGTGGCCTTCCCGGACTGGTTCATTGCCTCCTCCAAGAGAGACCAGCCCATCATTCTGACTTCAGAACTTGGGAAGTCATACAACACTGCCTTTGAATTAAATATA AATGACTAA,(SEQ ID NO 76) ATGTCAATGTGTAAACCTATTACTGGGACTATTAATGATTTGAATCAGCAAGTGTGGACCCTTCAGGGTCAGAACCTTGTGGCAGTTCCACGAAGTGACAGTGTGACCCCAGTCACTGTTGCTGTTATCACATGCAAGTATCCAGAGGCTCTTGAGCAAGGCAGAGGGGATCCCATTTATTTGGGAATCCAGAATCCAGAAATGTGTTTGTATTGTGAGAAGGTTGGAGAACAGCCCACATTGCAGCTAAAAGAGCAGAAGATCATGGATCTGTATGGCCAACCCGAGCCCGTGAAACCCTTCCTTTTCTACCGTGCCAAGACTGGTAGGACCTCCACCCTTGAGTCTGTGGCCTTCCCGGACTGGTTCATTGCCTCCTCCAAGAGAGACCAGCCCATCATTCTGACTTCAGAACTTGGGAAGTCATACAACACTGCCTTTGAATTAAATATAAATGACAGATCTGGCAGTTCTGACTACAAGGACGACGACGACAAGGGCAGTTCTCACCATCACCATCACCACTAG  and (SEQ ID NO 77)ATGATGTGTAAACCTATTACTGGGACTATTAATGATTTGAATCAGCAAGTGTGGACCCTTCAGGGTCAGAACCTTGTGGCAGTTCCACGAAGTGACAGTGTGACCCCAGTCACTGTTGCTGTTATCACATGCAAGTATCCAGAGGCTCTTGAGCAAGGCAGAGGGGATCCCATTTATTTGGGAATCCAGAATCCAGAAATGTGTTTGTATTGTGAGAAGGTTGGAGAACAGCCCACATTGCAGCTAAAAGAGCAGAAGATCATGGATCTGTATGGCCAACCCGAGCCCGTGAAACCCTTCCTTTTCTACCGTGCCAAGACTGGTAGGACCTCCACCCTTGAGTCTGTGGCCTTCCCGGACTGGTTCATTGCCTCCTCCAAGAGAGACCAGCCCATCATTCTGACTTCAGAACTTGGGAAGTCATACAACACTGCCTTTGAATTAAATATAAATGACAGATCTGGCAGTTCTGACTACAAGGACGACGACGACAAGGGCAGTTCTCACCATCACCATCACCACTAG.In another aspect, a host cell transfected or transduced with arecombinant vector that directs expression of a nucleic acid encoding anIL-1F9 polypeptide of the invention is provided. In another aspect, amethod of producing an isolated IL-1F9 polypeptide of the inventioncomprising culturing the host cell transfected or transduced with arecombinant vector that directs expression of an IL-1F9 polypeptide ofthe invention, under conditions promoting expression and isolating theexpressed IL-1F9 polypeptide is provided.

In another aspect, a composition, particularly a pharmaceuticalcomposition, comprising an IL-1F9 polypeptide of the invention and aphysiologically acceptable diluent, excipient or carrier is provided. Amethod of stimulating the immune system of an immunosuppresed subject,comprising administering the IL-1F9 polypeptide according of theinvention to an immunosuppresed subject in an amount sufficient tostimulate the subject's immune system is provided.

An antibody that specifically binds IL-1F9, where IL-1F9 may be a fulllength IL-1F9 or a truncant of full length IL-1F9, and preventsproteolytic cleavage thereof, particularly proteolytic cleavage to amore active form where activity is relative to that of the full lengthIL-1F9, is provided. An antibody that binds an IL-1F9 polypeptide of theinvention, and prevents signal transduction/activation through IL-1FRrp2is also provided. In some embodiments, an antibody binding an IL-1F9polypeptide selected from the group consisting of: SEQ ID NO 18, SEQ IDNO 19, SEQ ID NO 20 and SEQ ID NO 21, is provided. In some embodiments,the IL-1F9 antibody of the invention, is a monoclonal antibody,particularly a chimeric antibody, a humanized antibody or a fully humanantibody. The invention provides a composition, particularly apharmaceutical composition, comprising the IL-1F9 antibody of theinvention and a physiologically acceptable diluent, excipient orcarrier.

The invention also provides, a method of treating an inflammatory orautoimmune condition mediated by IL-1Rrp2, comprising administering theIL-1F9 antibody of the invention to a subject in an amount sufficient toameliorate at least one symptom mediated by IL-1Rrp2 of the condition.In one embodiment, the condition to be treated is an inflammatorycondition of the skin, lungs or airways mediated by IL-1Rrp2. In aparticular embodiment, the condition to be treated is selected from thegroup consisting of psoriasis, seborrheic dermatitis, atopic dermatitis,including chronic atopic dermatitis, allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris, nummular dermatitis,asthma, allergic rhinitis, gastro-esophageal reflux disease, arthriticconditions including, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis. In particular embodiments of the above methods, thesubject is a human.

In another aspect, the invention provides a method of identifying aprotease that cleaves an IL-1 family member comprising contacting asource of the protease with the IL-1 family member under conditionspromoting proteolytic cleavage of the IL-1 family member, anddetermining if the IL-1 family member has been proteolytically cleaved.The invention further provides a method of identifying an inhibitor of aprotease that cleaves an IL-1 family member comprising contacting theprotease with the IL-1 family member in the presence, and absence, of amolecule that is a potential inhibitor, under conditions promotingproteolytic cleavage of the IL-1 family member, and determining if theIL-1 family member has been proteolytically cleaved, wherein if the IL-1family member is not cleaved or is cleaved to a lesser degree in thepresence of the molecule, the molecule is an inhibitor.

DETAILED DESCRIPTION

The present invention provides compositions, kits, and methods relatingto members of the IL-1 family that require IL-1Rrp2 for signaling orinhibit signaling by competing with IL-1 family members that requireIL-1Rrp2 for signaling (hereinafter “IL-1Rrp2 requiring polypeptides”,for example, IL-1F5, F6, F8 and F9). Also provided are nucleic acids,and derivatives and fragments thereof, encoding such IL-1 familymembers. The invention further provides antigen binding proteins thatbind to these IL-1 family members. The provided methods include, forexample, methods of identifying and/or isolating a protease that cleavessuch an IL-1 family member, methods of making, identifying, or isolatingmolecules that modulate the interaction between an IL-1Rrp2-requiringIL-1 family member and a protease, methods of identifying other IL-1family members that interact with IL-1Rrp2 and methods of identifyingother IL-1R family members that interact with IL-1F5, F6, F8 and/or F9.

The invention also provides IL-1Rrp2 requiring polypeptides that havereproducibly high levels of biological activity as a result of theconformation of the amino terminal portion of the polypeptide. As shownin FIG. 1, there is a (Met or Ile)-Xaa-Asp sequence present in each ofthe IL-1 family members known to require IL-1Rrp2 for signaling (IL-1F5,F6, F8 and F9), which is marked by underlining of the Met/Ile and theAsp residues. An IL-1Rrp2 requiring polypeptide having an N-terminusnine residues upstream of the Met/Ile is highly active, whereas anIL-1Rrp2 requiring polypeptide extending further upstream is poorlyactive. IL-1Rrp2 requiring polypeptides that have an N-terminus at eightor seven residues upstream of the Met/Ile are poorly active or inactive.Moreover, the exact amino acid present at N-terminus of the IL-1Rrp2requiring polypeptides does not appear to be important. Severalpolypeptides were prepared, some of which had an N-terminal Metinitially, which N-terminal Met was cleaved off by an intracellularmethionyl aminopeptidase for some polypeptides; for others (those withbulky residues C-terminal to the Met) the aminopeptidase was not able toremove the methionine and for other polypeptides were prepared withoutan N-terminal Met as described in Example 1 below.

Polynucleotide and polypeptide sequences described herein are indicatedusing standard one- or three-letter abbreviations. Unless otherwiseindicated, each polypeptide sequence has an amino terminus at the leftand a carboxy terminus at the right; each single-stranded nucleic acidsequence, and the top strand of each double-stranded nucleic acidsequence, has a 5′ terminus at the left and a 3′ terminus at the right.A particular polypeptide or polynucleotide sequence also can bedescribed by explaining how it differs from a reference sequence.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, orsynthesized in a cellular system different from the cell from which itnaturally originates, will be “isolated” from its naturally associatedcomponents. A molecule also may be rendered substantially free ofnaturally associated components by isolation, using purificationtechniques well known in the art. Molecule purity or homogeneity may beassayed by a number of means well known in the art. For example, thepurity of a polypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

The terms “IL-1Rrp2 inhibitor” and “L-1Rrp2 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of IL-1Rrp2. Conversely, an “IL-1Rrp2 agonist” is amolecule that detectably increases at least one function of IL-1Rrp2.The inhibition caused by an IL-1Rrp2 inhibitor need not be complete solong as it is detectable using an assay. Any assay of a function ofIL-1Rrp2 can be used, examples of which are provided herein. Examples offunctions of IL-1Rrp2 that can be inhibited by an IL-1Rrp2 inhibitor, orcaused or increased by an IL-1Rrp2 agonist, include activation of NFkBsignaling pathways; activation of MAP kinases (Erk, INK, p38) and theirsignaling pathways; induction of cytokines; induction of chemokines;recruitment of neutrophils; enhancement of skin thickness (e.g.,induction of acanthosis and/or hyperkeratosis resembling that found inpsoriatic skin), downstream signaling, and so on. Examples of types ofIL-1Rrp2 inhibitors and IL-1Rrp2 agonists include, but are not limitedto, IL-1Rrp2 requiring polypeptides such as certain IL-1 family members(e.g., IL-1F6, F8 and F9, which are IL-1Rrp2 agonists, and IL-1F5, whichis an IL-1Rrp2 antagonist), antibodies, antibody fragments, and antibodyderivatives (for example, an antibody that binds an IL-1Rrp2 agonist andprevents proteolytic cleavage thereof).

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion as comparedto a corresponding full-length protein. Fragments can be, for example,at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100,150 or 200 amino acids in length. Fragments can also be, for example, atmost 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50,40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. A fragmentcan further comprise, at either or both of its ends, one or moreadditional amino acids, for example, a sequence of amino acids from adifferent naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence or a tag protein).

Polypeptides of the invention include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties. Analogs include muteins of a polypeptide. Forexample, single or multiple amino acid substitutions (e.g., conservativeamino acid substitutions) may be made in the naturally occurringsequence (e.g., in the portion of the polypeptide outside the domain(s)forming intermolecular contacts. A “conservative amino acidsubstitution” is one that does not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterize the parentsequence or are necessary for its functionality). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), whichare each incorporated herein by reference.

The present invention also provides non-peptide analogs ofIL-IL-1Rrp2-requiring polypeptides. Non-peptide analogs are commonlyused in the pharmaceutical industry as drugs with properties analogousto those of the template peptide. These types of non-peptide compoundare termed “peptide mimetics” or “peptidomimetics,” see, for example,Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which areincorporated herein by reference. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce anequivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a desired biochemical property orpharmacological activity), such as a human antibody, but have one ormore peptide linkages optionally replaced by a linkage selected from thegroup consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans),—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art.Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may also be used to generate more stable peptides. Inaddition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387(1992), incorporated herein by reference), for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

A “variant” of a polypeptide (e.g., an antibody) comprises an amino acidsequence wherein one or more amino acid residues are inserted into,deleted from and/or substituted into the amino acid sequence relative toanother polypeptide sequence. Variants of the invention include fusionproteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold with oneor more grafted complementarity determining regions (CDRs) or CDRderivatives. Such scaffolds include, but are not limited to,antibody-derived scaffolds comprising mutations introduced to, forexample, stabilize the three-dimensional structure of the antigenbinding protein as well as wholly synthetic scaffolds comprising, forexample, a biocompatible polymer. See, for example, Korndorfer et al.,2003, Proteins: Structure, Function, and Bioinformatics, Volume 53,Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Inaddition, peptide antibody mimetics (“PAMs”) can be used, as well asscaffolds based on antibody mimetics utilizing fibronection componentsas a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. From N-terminusto C-terminus, both light and heavy chains comprise the domains FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies often consist of less than about 10% antibody having specificbinding activity for the particular antigen. The percentage ofantigen-specific antibody can be increased by using multiplepurification steps. Antibodies that are enriched by affinitypurification using the antigen are often referred to as “monospecific.”

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and CDR fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies, triabodies, tetrabodies, and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to the polypeptide.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991. One or more CDRs may be incorporated into a moleculeeither covalently or noncovalently to make it an antigen bindingprotein. An antigen binding protein may incorporate the CDR(s) as partof a larger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRspermit the antigen binding protein to specifically bind to a particularantigen of interest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one example of a chimeric antibody, a portion ofthe heavy and/or light chain is identical with, homologous to, orderived from an antibody from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with, homologous to, or derived from anantibody (-ies) from another species or belonging to another antibodyclass or subclass. Also included are fragments of such antibodies thatexhibit the desired biological activity (i.e., the ability tospecifically bind an IL-1 family member). See, e.g., U.S. Pat. No.4,816,567 and Morrison, 1985, Science 229:1202-07.

A “neutralizing antibody” or “an inhibitory antibody” is an antibodythat inhibits the proteolytic activation of an IL-1 family member whenan excess of the anti-IL-1 family member antibody reduces the amount ofactivation by at least about 20% using an assay such as those describedherein in the Examples. In various embodiments, the antibody reduces theamount of amount of proteolytic activation of an IL-1 family member byat least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques well-known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, e.g., Bowie et al.,1991, Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g., ahuman IL-1 family member) if it binds to the antigen with a dissociationconstant of 1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof, of the invention.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293, 293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

IL-1Rrp2 Requiring IL-1 Family Members

In one aspect, the present invention provides proteins (e.g., IL-1family members, derivative, muteins and variants thereof) that requireIL-1Rrp2 for signaling, e.g., human IL-1 F5, F6, F8 and F9. The IL-1family members in accordance with the present invention include proteinsthat inhibit a biological activity of IL-1Rrp2, as well as proteins thatstimulate a biological activity of IL-1Rrp2. Examples of such biologicalactivities include activation of multiple kinase pathways, includingERK, p38MAPK, JNK, and IKK. In the skin, IL-1Rrp2 signaling can lead toan acanthotic, hyperkeratotic epidermis that resembles psoriatic skin,and in the lung it causes the recruitment of neutrophils.

Different IL-1 family members may utilize different domains of IL-1Rrp2for signaling, or act by different mechanisms of action. Examplesinclude but are not limited to proteins that cause signal transductionvia IL-1Rrp2, and proteins that inhibit signal transduction. The site ofaction may be, for example, intracellular (e.g., by interfering with anintracellular signaling cascade) or extracellular. An antagonisticprotein need not completely inhibit IL-1Rrp2 activity to find use in thepresent invention; rather, antagonistic proteins that reduce aparticular activity of IL-1Rrp2 are contemplated for use as well.Discussions herein of particular mechanisms of action for antagonisticproteins in treating particular diseases are illustrative only, and themethods presented herein are not bound thereby.

Other derivatives of the IL-1 family members within the scope of thisinvention include covalent or aggregative conjugates of the proteins, orfragments thereof, with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of an IL-1Rrp2requiring protein. For example, the conjugated peptide may be aheterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. IL-1Rrp2requiring protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of IL-1Rrp2 requiringpolypeptides (e.g., poly-His). An IL-1Rrp2 requiring polypeptides alsocan be linked to the FLAG® peptide (DYKDDDDK; SEQ ID NO:5), described inHopp et al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912.The FLAG peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody (mAb), enabling rapid assay andfacile purification of expressed recombinant protein. Reagents usefulfor preparing fusion proteins in which the FLAG peptide is fused to agiven polypeptide are commercially available (Sigma, St. Louis, Mo.).

Additional, useful tag proteins include green fluorescent protein (GFP;Chalfie et al., Science 263:802, 1994), an N-terminal peptide thatcontains recognition sites for a monoclonal antibody, a specificendopeptidase, and a site-specific protein kinase (PKA; Blanar andRutter, Science 256:1014, 1992), birA (Altman et al., Science 274:94,1996) and glutathione S transferase (GST: Smith and Johnson, Gene 67:31,1988).

Oligomers that contain one or more IL-1Rrp2 requiring proteins may beemployed as agonists or antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more IL-1Rrp2 requiring proteinsare contemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple IL-1Rrp2requiring proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the IL-1Rrp2 requiring proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of IL-1Rrp2 requiring proteins attached thereto,as described in more detail below.

In particular embodiments, the oligomers comprise from two to fourIL-1Rrp2 requiring proteins. The IL-1Rrp2 requiring proteins of theoligomer may be in any form, such as any of the forms described above,e.g., variants or fragments. Preferably, the oligomers comprise IL-1Rrp2requiring proteins that have activity (i.e., IL-1Rrp2 agonistic orantagonistic activity).

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins,” in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing an IL-1Rrp2 requiringpolypeptides or fragment thereof to the Fc region of an antibody. Thedimer can be made by, for example, inserting a gene fusion encoding thefusion protein into an appropriate expression vector, expressing thegene fusion in host cells transformed with the recombinant expressionvector, and allowing the expressed fusion protein to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed there from) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, an IL-1Rrp2 requiring polypeptide or fragmentthereof may be substituted for the variable portion of an antibody heavyand/or light chain.

Alternatively, the oligomer is a fusion protein comprising multipleIL-1Rrp2 requiring proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric IL-1Rrp2 requiring proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising an IL-1Rrp2 requiringprotein, fragment or derivative fused to a leucine zipper peptide isexpressed in suitable host cells, and the soluble oligomeric IL-1Rrp2requiring polypeptides fragments or derivatives that form are recoveredfrom the culture supernatant.

Antigen Binding Proteins

The present invention also provides antigen binding proteins that bindan IL-1Rrp2 requiring polypeptides (for example, an IL-1Rrp2 agonist orantagonist). Numerous types of antigen binding proteins and methods ofmaking them are known in the art. In one aspect, the present inventionprovides antigen binding proteins that interfere with the proteolyticactivation of an IL-1Rrp2 requiring polypeptides. Such antigen bindingproteins can be made against an IL-1 family member such as IL-1F6, F8 orF9 (or F5), or a fragment, variant or derivative thereof, and screenedin conventional assays for the ability to interfere with proteolyticactivation of the IL-1Rrp2 requiring protein.

In another aspect, the present invention includes an antigen bindingprotein that demonstrates species selectivity, and an antigen bindingprotein that has one or more of the following characteristics: binds toboth human and murine IL-1Rrp2 requiring protein, inhibits theproteolytic activation of human IL-1Rrp2 requiring protein, inhibits theproteolytic activation of murine IL-1Rrp2 requiring protein, binds to ornear the proteolytic cleavage site of IL-1Rrp2 requiring protein, causesrelatively little down-regulation of cell-surface expressed IL-1Rrp2.

Fragments of antigen binding proteins are also included. Antigen-bindingfragments of antigen binding proteins of the invention may be producedby conventional techniques. Examples of such fragments include, but arenot limited to, Fab and F(ab′)₂ fragments. Antibody fragments andderivatives produced by genetic engineering techniques also arecontemplated.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of non-human (e.g., murine) monoclonal antibodies. Suchhumanized antibodies may be prepared by known techniques, and offer theadvantage of reduced immunogenicity when the antibodies are administeredto humans.

Also included are human or partially human antibodies prepared innon-human animals (for example, mice in which one or more endogenousimmunoglobulin genes have been inactivated and replaced with humanimmunoglobulin). Antibodies produced in the animal incorporate humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal.

In another aspect, the present invention provides monoclonal antibodiesthat bind to IL-1Rrp2 requiring protein(s). Monoclonal antibodies may beproduced using any technique known in the art, e.g., by immortalizingspleen cells harvested from the transgenic animal after completion ofthe immunization schedule. The spleen cells can be immortalized usingany technique known in the art, e.g., by fusing them with myeloma cellsto produce hybridomas.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block an IL-1Rrp2 induced activity. Examples of such screensare provided in the examples below.

Molecular evolution of the complementarity determining regions (CDRs) inthe center of the antibody binding site also has been used to isolateantibodies with increased affinity, for example, antibodies havingincreased affinity for c-erbB-2, as described by Schier et al., 1996, J.Mol. Biol. 263:551. Accordingly, such techniques are useful in preparingantibodies to IL-1Rrp2 requiring proteins.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

In one aspect, the present invention provides antigen-binding fragmentsof an anti-IL-1Rrp2 requiring polypeptides antibody of the invention.Such fragments can consist entirely of antibody-derived sequences or cancomprise additional sequences. Examples of antigen-binding fragmentsinclude Fab, F(ab′)2, single chain antibodies, diabodies, triabodies,tetrabodies, and domain antibodies. Other examples are provided in Lundeet al., 2002, Biochem. Soc. Trans. 30:500-06.

In another aspect, the present invention provides an antigen bindingprotein that binds at or near the protease cleavage site of humanIL-1F6, F8 or F9, or IL-1F5. Antigen binding proteins that bind to theprotease cleavage site can be made using any technique known in the art.For example, such antigen binding proteins can be isolated using thefull-length an IL-1Rrp2 requiring protein, or a smaller fragment thereofcomprising or consisting of the protease cleavage site (examples ofwhich are provided herein). Antigen binding proteins so isolated can bescreened to determine their binding specificity using any method knownin the art (examples of which are provided herein). Such antigen bindingproteins that function as IL-1Rrp2 antagonists may be employed intreating any IL-1Rrp2-induced condition, including but not limited toinflammatory conditions.

The present invention further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of an IL-1Rrp2requiring protein, or to an epitope of one IL-1Rrp2 requiringpolypeptide and an epitope of another IL-1Rrp2 requiring protein, viatwo different antigen binding sites or regions. Numerous methods ofpreparing bispecific antibodies are known in the art.

Although human, partially human, or humanized antibodies will besuitable for many applications, particularly those involvingadministration of the antibody to a human subject, other types ofantigen binding proteins will be suitable for certain applications. Thenon-human antibodies of the invention can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomologous or rhesusmonkey) or ape (e.g., chimpanzee)). Non-human antibodies of theinvention can be used, for example, in in vitro and cell-culture basedapplications, or any other application where an immune response to theantibody of the invention does not occur, is insignificant, can beprevented, is not a concern, or is desired

Peptibodies

An additional class of compounds useful in the practice of the methodsof the present invention are compounds sometimes referred to aspeptibodies. Such compounds are biologically active peptides having anincreased in vivo half-life and reduced immunogenicity profile. This isaccomplished by fusion of the peptide(s) with a vehicle, as described inU.S. Pat. No. 6,660,843 (the disclosure of which is incorporated byreference herein). Briefly, pharmacologically active compounds areprepared selecting at least one peptide that modulates the activity of aprotein of interest, e.g., in this case selecting a peptide thatantagonizes the activity of an IL-1Rrp2 requiring polypeptide (e.g.,IL-1F6, F8, F9, or IL-1F5), as in a peptide that inhibits proteolyticcleavage of an IL-1Rrp2 agonist; and preparing a fusion protein of theselected peptide and multimerizing vehicle

One such vehicle is an Fc domain. The peptides screened as describedabove are expressed in a phage display library. The vehicle and thepeptide may be linked through the N- or C-terminus of the peptide or thevehicle, as described further in U.S. Pat. No. 6,660,843. Derivatives ofthe above compounds are also encompassed by this invention.

Antagonistic molecules useful in the processes of this invention may beprepared by standard synthetic methods, recombinant DNA techniques, orany other methods of preparing peptides and fusion proteins. Compoundsof this invention that encompass non-peptide portions may be synthesizedby standard organic chemistry reactions, in addition to standard peptidechemistry reactions when applicable.

Peptibodies may be used to prepare derivative and other forms thereof,substantially as described for antibodies.

Nucleic Acids

In one aspect, the present invention provides isolated nucleic acidmolecules. The nucleic acids comprise, for example, polynucleotides thatencode all or part of an IL-1Rrp2 requiring polypeptide, for example,IL-1F5, F6, F8 or F9, one or both chains of an antibody of theinvention, or a fragment, derivative, mutein, or variant thereof,polynucleotides sufficient for use as hybridization probes, PCR primersor sequencing primers for identifying, analyzing, mutating or amplifyinga polynucleotide encoding a polypeptide, anti-sense nucleic acids forinhibiting expression of a polynucleotide, and complementary sequencesof the foregoing. The nucleic acids can be any length. They can be, forexample, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 ormore nucleotides in length, and/or can comprise one or more additionalsequences, for example, regulatory sequences, and/or be part of a largernucleic acid, for example, a vector. The nucleic acids can besingle-stranded or double-stranded and can comprise RNA and/or DNAnucleotides, and artificial variants thereof (e.g., peptide nucleicacids).

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anIL-1Rrp2 requiring polypeptides) that it encodes. Mutations can beintroduced using any technique known in the art. In one embodiment, oneor more particular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property (e.g., binding to IL-1Rrp2or blocking the proteolytic activation of an IL-1 family member such asIL-1F5, F6, F8 or F9).

In another aspect, the present invention provides vectors comprising anucleic acid encoding a polypeptide of the invention or a portionthereof. Examples of vectors include, but are not limited to, plasmids,viral vectors, non-episomal mammalian vectors and expression vectors,for example, recombinant expression vectors.

The recombinant expression vectors of the invention can comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. The recombinant expression vectors includeone or more regulatory sequences, selected on the basis of the hostcells to be used for expression, which is operably linked to the nucleicacid sequence to be expressed. Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoterand cytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

In another aspect, the present invention provides host cells into whicha recombinant expression vector of the invention has been introduced. Ahost cell can be any prokaryotic cell (for example, E. coli) oreukaryotic cell (for example, yeast, insect, or mammalian cells (e.g.,CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryoticcells via conventional transformation or transfection techniques. Forstable transfection of mammalian cells, it is known that, depending uponthe expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die), among other methods.

Expression of Recombinant Proteins or Polypeptides

Any expression system known in the art can be used to make therecombinant polypeptides of the invention (i.e., recombinant IL-1Rrp2polypeptides, recombinant antigen binding proteins, peptibodies and thelike). In general, host cells are transformed with a recombinantexpression vector that comprises DNA encoding a desired polypeptide.Among the host cells that may be employed are prokaryotes, yeast orhigher eukaryotic cells. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include insect cells and established cell lines of mammalianorigin. Examples of suitable mammalian host cell lines include the COS-7line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163),Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) celllines, and the CVI/EBNA cell line derived from the African green monkeykidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991,EMBO J. 10:2821. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described byPouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NewYork, 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., in the caseof an antigen binding protein, over a matrix having all or a portion ofthe antigen (e.g., an IL-1Rrp2 requiring polypeptides or portionthereof) bound thereto, or in the case of an IL-1Rrp2 requiring protein,over a matrix having all or a portion of IL-1Rrp2, or an antigen bindingprotein that binds the IL-1Rrp2 requiring protein bound thereto.Polypeptides or proteins contemplated for use herein includesubstantially homogeneous recombinant mammalian IL-1Rrp2 requiringpolypeptides, and/or antibodies thereto, substantially free ofcontaminating endogenous materials.

The proteins may be prepared, and screened for desired properties, byany of a number of known techniques. Certain of the techniques involveisolating a nucleic acid encoding a polypeptide chain (or portionthereof) of an IL-1Rrp2 requiring polypeptides of interest (e.g.,IL-1F6, F8 or F (or IL-1F5), and manipulating the nucleic acid throughrecombinant DNA technology. Nucleic acids encoding antigen bindingproteins that bind an IL-1Rrp2 requiring polypeptides can be similarlymanipulated. The nucleic acid may be fused to another nucleic acid ofinterest, or altered (e.g., by mutagenesis or other conventionaltechniques) to add, delete, or substitute one or more amino acidresidues, for example.

Protease Isolation and Assay

The IL-1Rrp2 requiring proteins described herein will also be useful inidentifying and/or isolating a protease or proteases that cleave theIL-1Rrp-2 requiring proteins to yield the bioactive form thereof. Usefulmethods are described, for example, in the Handbook Of ProteolyticEnzymes, Second Edition, edited by A. Barrett, N. Rawlings and J.Woessner (Academic Press, 2004). In general, methods for identifying theprotease(s) might include (a) testing known proteases for their abilityto generate biologically active material; (b) screening cellsupernatants, membranes, or lysates for their ability to generatebiologically active material; and (c) using the full-length (uncleaved)IL-1Rrp2 requiring protein as an affinity reagent to purify theprotease(s).

For example, known proteases can be tested for the ability to cleave anIL-1Rrp2 requiring protein into bioactive form by contacting a knownprotease with the IL-1Rrp2 requiring protein under conditions promotingprotease activity, than determining what effect, if any, the proteasehad upon the biological activity of the IL-1Rrp2 requiring polypeptides(for example, activity as an agonist or an antagonist as describedherein). Cells that express a protease (or proteases) that cleaveIL-1Rrp2 requiring proteins, either intracellularly, as a solublepolypeptide or as a cell-surface associated protein, can be identifiedin a similar manner. It may further be possible to use an IL-1Rrp2requiring protein to select a cell population enriched for proteaseexpression by using a panning or cell sorting technique, many of whichare known in the art. When a cellular source of the protease has beenidentified, the IL-1Rrp2 requiring protein(s) can be used in an effortto isolate the protease.

Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for activity within ashort amount of time. These methods include homogeneous assay formatssuch as fluorescence resonance energy transfer, fluorescencepolarization, time-resolved fluorescence resonance energy transfer,scintillation proximity assays, reporter gene assays, fluorescencequenched enzyme substrate, chromogenic enzyme substrate andelectrochemiluminescence.

One such assay is based on fluorescence resonance energy transfer (FRET;for example, HTRF®, Packard Instrument Company, Meriden, Conn.; LANCE™,PerkinElmer LifeSciences, Wallac Oy., Turku, Finland) between twofluorescent labels, an energy donating long-lived chelate label and ashort-lived organic acceptor. The energy transfer occurs when the twolabels are brought in close proximity via the molecular interactionbetween an IL-1Rrp2 polypeptide and a protease that cleaves it.

Indications

In one aspect, the present invention provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, relieve, orameliorate (“treat”) a disease, disorder, condition, or illness (“acondition”). Among the conditions to be treated in accordance with thepresent invention are conditions characterized by inappropriateexpression or activity of IL-1Rrp2 and/or agonists or antagoniststhereof (e.g., IL-1F6, F8 and/or F9 for the former, IL-1F5 for thelatter). In some such conditions, the expression or activity level ofthe receptor or agonist(s) thereof is too high; in other cases theexpression or activity level of an antagonist(s) thereof is too low.Treatment comprises administering an IL-1Rrp2 antagonist as describedherein.

Conditions that fall within this category often exhibit an inflammatoryskin phenotype with characteristics common to those seen in humanpsoriatic skin. Such characteristic include acanthosis, hyperkeratosis,dermal infiltrate, increased expression of keratin 6, increasedexpression of keratin 14, increased expression of ICAM-1 in dermis,basal keratinocytes and blood vessels, increased expression ofmacrophage marker BM8 in dermis and decreased expression in epidermis,and decreased expression of T cell marker CD3 in epidermis.

Specific medical conditions and diseases that are treatable orpreventable with the IL-1Rrp2 antagonists of this invention includethose associated with inflammatory skin diseases including, but notlimited to psoriasis, seborrheic dermatitis, atopic dermatitis(including chronic atopic dermatitis or CAD), allergic contactdermatitis, lichen simplex chronicus, pityriasis rubra pilaris andnummular dermatitis.

Moreover, normal airway epithelium has relatively high expression ofIL-1 family members F5, F6, F8 and F9, as well as the receptor IL-1Rrp2,and intranasal instillation of F8 or F9 leads to an influx ofneutrophils into the lung. Accordingly IL-1Rrp2 antagonists may beindicated for inflammatory conditions of the airway, for example asthmaand allergic rhinitis. IL-1F9 and IL-1Rrp2 are highly expressed in theesophagus, and the interaction between this cytokine/receptor pair mayplay a role in gastro-esophageal reflux disease (GERD), which mayaccordingly be ameliorated by IL-1Rrp2 antagonists.

IL-1F8 and IL-1Rrp2 are also expressed in synovialfibroblasts andchondrocytes, and are induced to higher levels in those cells by IL-1and TNF. Moreover, these cells respond to exogenous IL-1F8 bysynthesizing IL-6, IL-8 and nitric oxide. Accordingly, the IL-1Rrp2antagonists of the invention may have use in arthritic conditionsmediated by the induced polypeptides (i.e., rheumatoid arthritis,psoriatic arthritis, other arthritic conditions in which TNF and/or IL-1play a role, and osteoarthritis and related conditions in which nitricoxide plays a role)

The methods described herein can be treated with the IL-1Rrp2antagonists of this invention in combination with other cytokines,cytokine inhibitors and reagents (also referred to herein asimmunomodulators). For example, IL-18 antagonists; including solubleIL-18 receptor, antibodies to IL-18 or the IL-18 receptor, IL-18 bindingprotein; TNF inhibitors, including ENBREL®; IL-1 inhibitors, includingsoluble forms of type II IL-1R, type II IL-1R, antibodies to IL-1,antibodies to type I IL-1R; and or other active agents that areeffective in treating the disclosed medical conditions and diseases.

The compositions and/or methods of the present invention also can beused, for example, in cosmetic treatments, in veterinary treatments, toincrease longevity, to treat reproductive defects, and to treat avariety of IL-1Rrp2 related disorders. In addition, in certain suchconditions, the expression or activity level of IL-1Rrp2 agonists is toolow, and the treatment comprises administering an IL-1Rrp2 agonist suchas IL-1F6, F8 and/or F9; such treatments are also comprehended herein.

Therapeutic Methods and Administration of IL-1Rrp2 Antagonists

Certain methods provided herein comprise administering an IL-1Rrp2antagonist to a subject, thereby reducing an IL-1 family member-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous IL-1Rrp2-expressing cells with an IL-1Rrp2 antagonist, e.g.,via administration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An IL-1Rrp2 antagonist need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient an IL-1Rrp2 antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the molecules of the invention are administered to a subjectin a manner appropriate to the indication. Pharmaceutical compositionsmay be administered by any suitable technique, including but not limitedto parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of IL-1Rrp2 antagonist s thereto in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an IL-1Rrp2 requiring polypeptides that binds anenzyme such as a protease ex vivo. The IL-1Rrp2 requiring polypeptidesmay be bound to a suitable insoluble matrix or solid support material.

Advantageously, IL-1Rrp2 antagonist are administered in the form of acomposition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to an IL-1Rrp2antagonist.

In one embodiment, the pharmaceutical composition comprise an IL-1Rrp2antagonist of the invention together with one or more substancesselected from the group consisting of a buffer, an antioxidant such asascorbic acid, a low molecular weight polypeptide (such as those havingfewer than 10 amino acids), a protein, an amino acid, a carbohydratesuch as glucose, sucrose or dextrins, a chelating agent such as EDTA,glutathione, a stabilizer, and an excipient. Neutral buffered saline orsaline mixed with conspecific serum albumin are examples of appropriatediluents. In accordance with appropriate industry standards,preservatives such as benzyl alcohol may also be added. The compositionmay be formulated as a lyophilizate using appropriate excipientsolutions (e.g., sucrose) as diluents. Suitable components are nontoxicto recipients at the dosages and concentrations employed. Furtherexamples of components that may be employed in pharmaceuticalformulations are presented in Remington's Pharmaceutical Sciences,16^(th) Ed. (1980) and 20^(th) Ed. (2000), Mack Publishing Company,Easton, Pa.

Kits for use by medical practitioners include an IL-1Rrp2-inhibitingsubstance of the invention and a label or other instructions for use intreating any of the conditions discussed herein. In one embodiment, thekit includes a sterile preparation of one or more IL-1Rrp2 antagonist sthereto, which may be in the form of a composition as disclosed above,and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular IL-1Rrp2antagonists employed, the nature and severity of the disease to betreated, whether the condition is acute or chronic, and the size andgeneral condition of the subject. Appropriate dosages can be determinedby procedures known in the pertinent art, e.g. in clinical trials thatmay involve dose escalation studies.

A IL-1Rrp2-inhibiting substance of the invention may be administered,for example, once or more than once, e.g., at regular intervals over aperiod of time. In particular embodiments, an IL-1Rrp2 antagonist isadministered over a period of at least a month or more, e.g., for one,two, or three months or even indefinitely. For treating chronicconditions, long-term treatment is generally most effective. However,for treating acute conditions, administration for shorter periods, e.g.from one to six weeks, may be sufficient. In general, the IL-1Rrp2antagonist is administered until the patient manifests a medicallyrelevant degree of improvement over baseline for the chosen indicator orindicators.

Particular embodiments of the present invention involve administering anIL-1Rrp2 antagonist at a dosage of from about 1 ng of protein per kg ofsubject's weight per day (“1 ng/kg/day”) to about 10 mg/kg/day, morepreferably from about 500 ng/kg/day to about 5 mg/kg/day, and mostpreferably from about 5 micrograms/kg/day to about 2 mg/kg/day, to asubject. In additional embodiments, an IL-1Rrp2 antagonist isadministered to adults one time per week, two times per week, or threeor more times per week, to treat an IL-1Rrp2-mediated disease, conditionor disorder, e.g., a medical disorder disclosed herein. If injected, theeffective amount of IL-1Rrp2 antagonist per adult dose may range from1-20 mg/m², and preferably is about 5-12 mg/m². Alternatively, a flatdose may be administered; the amount may range from 5-100 mg/dose. Onerange for a flat dose is about 20-30 mg per dose. In one embodiment ofthe invention, a flat dose of 25 mg/dose is repeatedly administered byinjection. If a route of administration other than injection is used,the dose is appropriately adjusted in accordance with standard medicalpractices. One example of a therapeutic regimen involves injecting adose of about 20-30 mg of IL-1Rrp2 antagonist to one to three times perweek over a period of at least three weeks, though treatment for longerperiods may be necessary to induce the desired degree of improvement.For pediatric subjects (age 4-17), one exemplary suitable regimeninvolves the subcutaneous injection of 0.4 mg/kg, up to a maximum doseof 25 mg of IL-1Rrp2 antagonist administered two or three times perweek.

Particular embodiments of the methods provided herein involvesubcutaneous injection of from 0.5 mg to 10 mg, preferably from 3 to 5mg, of an IL-1Rrp2 antagonist, once or twice per week. Anotherembodiment is directed to pulmonary administration (e.g., by nebulizer)of 3 or more mg of IL-1Rrp2 antagonist once a week.

Examples of therapeutic regimens provided herein comprise subcutaneousinjection of an IL-1Rrp2 antagonist once a week, at a dose of 1.5 to 3mg, to treat a condition in which IL-1Rrp2 signaling plays a role.Examples of such conditions are provided herein and include, forexample, inflammatory conditions of the skin, including, but not limitedto psoriasis, seborrheic dermatitis, atopic dermatitis (includingchronic atopic dermatitis or CAD), allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris and nummular dermatitis.Weekly administration of IL-1Rrp2 antagonist is continued until adesired result is achieved, e.g., the subject's symptoms subside.Treatment may resume as needed, or, alternatively, maintenance doses maybe administered.

Other examples of therapeutic regimens provided herein comprisesubcutaneous or intravenous administration of a dose of 1, 3, 5, 6, 7,8, 9, 10, 11, 12, 15, or 20 milligrams of an IL-1Rrp2 inhibitor of thepresent invention per kilogram body mass of the subject (mg/kg). Thedose can be administered once to the subject, or more than once at acertain interval, for example, once a day, three times a week, twice aweek, once a week, three times a month, twice a month, once a month,once every two months, once every three months, once every six months,or once a year. The duration of the treatment, and any changes to thedose and/or frequency of treatment, can be altered or varied during thecourse of treatment in order to meet the particular needs of thesubject.

In another embodiment, an IL-1Rrp2 antagonist is administered to thesubject in an amount and for a time sufficient to induce an improvement,preferably a sustained improvement, in at least one indicator thatreflects the severity of the disorder that is being treated. Variousindicators that reflect the extent of the subject's illness, disease orcondition may be assessed for determining whether the amount and time ofthe treatment is sufficient. Such indicators include, for example,clinically recognized indicators of disease severity, symptoms, ormanifestations of the disorder in question. In one embodiment, animprovement is considered to be sustained if the subject exhibits theimprovement on at least two occasions separated by two to four weeks.The degree of improvement generally is determined by a physician, whomay make this determination based on signs, symptoms, biopsies, or othertest results, and who may also employ questionnaires that areadministered to the subject, such as quality-of-life questionnairesdeveloped for a given disease.

Elevated levels of IL-1Rrp2, IL-1F6, IL-1F8 and/or IL-1F9 and/oractivation of thereof, and/or decreased levels and/or activation ofIL-1F5, are associated with a number of disorders, including, forexample, inflammatory conditions of the skin, including, but not limitedto psoriasis, seborrheic dermatitis, atopic dermatitis (includingchronic atopic dermatitis or CAD), allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris and nummular dermatitis.Other such conditions include inflammatory conditions of the airway, ofthe esophagus, and the joints.

Subjects with a given disorder may be screened, to identify thoseindividuals who have elevated IL-1Rrp2, IL-1F6, IL-1F8 and/or IL-1F9activation (or decreased IL-1F5 activation), thereby identifying thesubjects who may benefit most from treatment with an IL-1Rrp2antagonist. Thus, treatment methods provided herein optionally comprisea first step of measuring a subject's IL-1Rrp2, IL-1F6, IL-1F8 and/orIL-1F9 (or IL-1F5) activation levels. An IL-1Rrp2 antagonist may beadministered to a subject in whom IL-1Rrp2, IL-1F6, IL-1F8 and/or IL-1F9activation is elevated above normal, and/or whose IL-1F5 activity isbelow normal.

A subject's levels of IL-1Rrp2, IL-1F6, IL-1F8, IL-1F9 and/or IL-1F5activity may be monitored before, during and/or after treatment with anIL-1Rrp2 antagonist, to detect changes, if any, in IL-1Rrp2, IL-1F6,IL-1F8, IL-1F9 and/or IL-1F5 activity. For some disorders, the incidenceof elevated IL-1Rrp2, IL-1F6, IL-1F8 and/or IL-1F9 activity, ordecreased IL-1F5 activity, may vary according to such factors as thestage of the disease or the particular form of the disease. Knowntechniques may be employed for measuring IL-1Rrp2, IL-1F6, IL-1F8,IL-1F9 and/or IL-1F5 activity, e.g., in a subject's serum, blood ortissue samples. IL-1Rrp2, IL-1F6, IL-1F8, IL-1F9 and/or IL-1F5 activitymay be measured using any suitable technique.

Particular embodiments of methods and compositions of the inventioninvolve the use of an IL-1Rrp2 antagonist and one or more additionalIL-1Rrp2 antagonists, for example, two or more IL-1Rrp2 requiringproteins of the invention, two or more antigen binding proteins of theinvention, or combinations of IL-1Rrp2 requiring proteins and antigenbinding proteins. In further embodiments, IL-1Rrp2 antagonists areadministered alone or in combination with other agents useful fortreating the condition with which the patient is afflicted. Examples ofsuch agents include both proteinaceous and non-proteinaceous drugs. Whenmultiple therapeutics are co-administered, dosages may be adjustedaccordingly, as is recognized in the pertinent art. “Co-administration”and combination therapy are not limited to simultaneous administration,but also include treatment regimens in which an IL-1Rrp2 antagonist isadministered at least once during a course of treatment that involvesadministering at least one other therapeutic agent to the patient.

Examples of other agents that may be co-administered with an IL-1Rrp2antagonist are other IL-1Rrp2 requiring proteins or antigen bindingproteins, or therapeutic polypeptides that are chosen according to theparticular condition to be treated. Alternatively, non-proteinaceousdrugs that are useful in treating one of the particular conditionsdiscussed above may be co-administered with IL-1Rrp2 antagonist.

Combination Therapy

In another aspect, the present invention provides a method of treating asubject with an IL-1Rrp2 antagonist, and one or more other treatments.In one embodiment, such a combination therapy achieves synergy or anadditive effect by, for example, attacking multiple sites or moleculartargets in a tumor. Types of combination therapies that can be used inconnection with the present invention include inhibiting or activating(as appropriate) multiple nodes in a single disease-related pathway,multiple pathways in a target cell, and multiple cell types within atarget tissue.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of theagonists or antagonists described herein. In another embodiment, themethod comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)IL-1Rrp2-mediated signal transduction. Examples of such methods includeusing combinations of two or more IL-1Rrp2 requiring proteins and/orantigen binding proteins, an IL-1Rrp2 requiring polypeptide or antigenbinding protein and one or more other therapeutic moiety havinganti-inflammatory properties (for example, non-steroidalanti-inflammatory agents, steroids, and/or immunomodulators), or anIL-1Rrp2 requiring polypeptide or antigen binding protein and one ormore other treatments (e.g., surgery, ultrasound, or treatment effectiveto reduce inflammation). Furthermore, one or more IL-1Rrp2 antagonistcan be used in combination with one or more molecules or othertreatments, wherein the other molecule(s) and/or treatment(s) do notdirectly bind to or affect IL-1Rrp2, but which combination is effectivefor treating or preventing the condition being treated. In oneembodiment, one or more of the molecule(s) and/or treatment(s) treats orprevents a condition that is caused by one or more of the othermolecule(s) or treatment(s) in the course of therapy, e.g., nausea,fatigue, alopecia, cachexia, insomnia, etc. In every case where acombination of molecules and/or other treatments is used, the individualmolecule(s) and/or treatment(s) can be administered in any order, overany length of time, which is effective, e.g., simultaneously,consecutively, or alternately. In one embodiment, the method oftreatment comprises completing a first course of treatment with onemolecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe IL-1Rrp2 antagonists described herein and one or more othertreatments (e.g., a therapeutic or palliative treatment). Where a methodcomprises administering more than one treatment to a subject, it is tobe understood that the order, timing, number, concentration, and volumeof the administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, which arehereby incorporated by reference. The following examples, both actualand prophetic, are provided for the purpose of illustrating specificembodiments or features of the instant invention and do not limit itsscope.

Example 1 Example 1A Preparation of IL-1F6 Variants

This example describes the preparation of various N-terminal variants ofIL-1F6. The various N-terminal deletion variants are shown in Table 1below:

TABLE 1 Amino Acid Sequence of IL-1F6 Variants IL-1 F6 VariantPolypeptides: (Hu indicates human; WT indicates full length wild typepolypeptide, FpH indicates a FLAG poly His tag., the bold,italicized, upper case letter and number refers to the indicated aminoN-Terminal amino acid sequence of IL-1F6 acid and its position inVariants ( . . . indicates intervening SEQ ID NO 2 whereamino acids of human IL-1F6 (SEQ ID NO the position is relative SEQ2; FLAG indicates the amino acid sequence to the N-Terminal IDDYKDDDDK; polyHis indicates a chain of amino acid at position NO:multiple histidines, typically about 6) one of SEQ ID NO 2.) 22MEKALKIDTPQQGSIQDI...FGLTMLF-FLAG-polyHis HuIL-1F6WT/FpH 23    MKIDTPQQGSIQDI...FGLTMLF-FLAG-polyHis HuIL-1F6

/FpH 24      MIDTPQQGSIQDI...FGLTMLF-FLAG-polyHis HuIL-1F6

/FpH 25       MDTPQQGSIQDI...FGLTMLF-FLAG-polyHis HuIL-1F6

/FpH 26      KIDTPQQGSIQDI...FGLTMLF HuIL-1F6

Note: The IL1-F6 polypeptide occurs in 2 isotypes. One isotype comprisesa Q at amino acid 12, relative to the M a position one of the wild typeIL-1F6 indicated in Table 1 as SEQ ID NO 22 and another that encodes Rat position 12. Variants of both isotypes are provided by thecompositions and methods of the invention.

The N-terminal variants that comprise a FLAG-polyHis C-terminal tag wereprepared as described immediately below. N-terminal variants wereamplified via PCR using as a template a cDNA clone encoding huIL-1F6(AF201831). An N-terminal methionine was placed directly before thedesired starting amino acid of each variant for proper translationalinitiation. FLAG® (Sigma-Aldrich, St. Louis, Mo.) and polyHis tags wereadded to the C-terminus for purification/detection. NdeI and XhoI siteswere added to the 5′ and 3′ ends, respectively. The resulting ampliconswere digested with NdeI and XhoI and subcloned into the E. coliexpression vector, pAMG21 (ATCC #98113). The resulting constructs wereintroduced into E. coli DH10B and expression was induced by the additionof [N-(3-oxo-hexanoyl) homoserine lactone]. The expressed polypeptideswere then purified from bacterial lysates (soluble fraction) by affinitychromatography using Ni-NTA columns (Qiagen, Germantown, Md. Cat #30600)as per manufacturer's protocol, and tested for activity in a reporterassay substantially as described in Example 2. Results are shown inTable 5 below.

The N-terminal variant, HuIL-1F6K6, was prepared by PCR amplifying atemplate cDNA clone encoding huIL-1F6K6 variant. The primers used in PCRallowed the PCR products to be placed using the In-Fusion™ PCR cloningsystem (Clontech, Mountain View, Calif., cat#631774), in accordance withmanufacturer's protocol, into a pET-SUMO vector (Invitrogen, Carlsbad,Calif., cat K300-01). This procedure put a pH-SUMO tag 5′ to the desiredstarting amino acid of the IL-1F6 variant in the vector resulting in thefollowing nucleic acid sequence where the pH SUMO tag runs from the 5′end to the underlined nucleotide in bold which indicates the start ofthe nucleic acid sequence encoding HuIL-1F6K6:

(SEQ ID NO 78) ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCCTGGTGCCGCGCGGCAGCGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGTAAAATTGACACACCTCAGCGGGGGAGCATTCAGGATATCAATCATCGGGTGTGGGTTCTTCAGGACCAGACGCTCATAGCAGTCCCGAGGAAGGACCGTATGTCTCCAGTCACTATTGCCTTAATCTCATGCCGACATGTGGAGACCCTTGAGAAAGACAGAGGGAACCCCATCTACCTGGGCCTGAATGGACTCAATCTCTGCCTGATGTGTGCTAAAGTCGGGGACCAGCCCACACTGCAGCTGAAGGAAAAGGATATAATGGATTTGTACAACCAACCCGAGCCTGTGAAGTCCTTTCTCTTCTACCACAGCCAGAGTGGCAGGAACTCCACCTTCGAGTCTGTGGCTTTCCCTGGCTGGTTCATCGCTGTCAGCTCTGAAGGAGGCTGTCCTCTCATCCTTACCCAAGAACTGGGGAAAGCCAACACTACTGACTTTGG GTTAACTATGCTGTTTTAA.

The resulting construct was introduced into E. coli DH10B. Expressionwas carried out using Overnight Express™ Autoinduction System (Novagen,Darmstadt, Germany, cat#71300-3) per manufacturer's protocol. The E.Coli cells were centrifuged and frozen.

Frozen E. coli cell pellets were thawed in TBS made in Roche CompleteEDTA free protease inhibitor cocktail. After thawing, benzonase wasadded. Cells were lysed via passage through a Microfluidicsmicrofluidizer 110L device (MFIC Corporation, Newton, Mass.) andresulting lysates clarified via centrifugation. Supernatants weresterile filtered and loaded onto 5 mL HisTrap (GE Biosciences,Piscataway, N.J.) Ni-sepharose columns equilibrated in Tris, NaCl,imidazole pH 7.4. Columns were washed with imidazole in TBS. Protein waseluted with a imidazole linear gradient. Eluted fractions were pooledand dialyzed into PBS using MWCO Slide-A-Lyzers (Pierce Biotechnology,Inc., Rockland, Ill.). LifeSensors SUMO Protease-1 was used to cleavethe His-SUMO fusion partner from IL-1F6 variant. Cleavage reactionproducts were dialyzed into TBS containing EDTA, pH 7.4 using MWCOSlide-A-Lyzers. Dialyzed pools were then passed back over HisTrapNi-Sepharose columns and flow-through fractions containing pureliberated IL-1F6 variant were retained. and tested for activity asdescribed in Example 2B below. Results are shown in Table 7 below.

Example 1B Preparation of IL-1F8 Variants

Variants of IL-1F8 comprising a FLAG-polyHis tag were prepared in asubstantially similar manner as for IL-F6 FLAG-polyHis tag variantsdescribed in Example 1A above, using as a template a cDNA clone encodinghuIL-1F8 (AF201833). Likewise, HuIL-1F8R5 (lacking a FLAG-polyHis tag),using as a template a cDNA clone encoding huIL-1F8 (AF201833), wasprepared and purified in a substantially similar manner to HuIL-1F6K6described in Example 1A above. The various N-terminal deletion variantsare shown in Table 2 below:

TABLE 2 Amino Acid Sequence of IL-1F8 Variants IL-1F8 VariantPolypeptide: (Hu indicates human; WT indicates full length wild typepolypeptide, FpH indicates a FLAG poly His tag., hebold, italicized, upper case letter and number refers to the indicatedN-Terminal amino acid sequence of IL-1F8 amino acid and itsVariants (... indicates intervening amino position in SEQ IDacids of human IL-1F8 (SEQ ID NO 3); ( )  NO 3 where theindicates that the enclosed amino acid is  position is relative toremoved; FLAG  indicates the amino acid the N-Terminal amino SEQ IDsequence DYKDDDDK; polyHis indicates a acid at position one of NO:chain of multiple histidines, typically 6) SEQ ID NO 3.) 27MNPQREAAPKSYAIR...FYLDSVE-FLAG-polyHis HuIL-1F8WT/FpH 28  MQREAAPKSYAIR...FYLDSVE-FLAG-polyHis HuIL-1F8

/FpH 29    MREAAPKSYAIR...FYLDSVE-FLAG-polyHis HuIL-1F8

/FpH 30     MEAAPKSYAIR...FYLDSVE-FLAG-polyHis HuIL-1F8

/FpH 31    (M)AAPKSYAIR...FYLDSVE-FLAG-polyHis HuIL-1F8

/FpH 32     REAAPKSYAIR...FYLDSVE HuIL-1F8R5

The FLAG-polyHis constructs were expressed in E. coli, and the expressedpolypeptides purified from bacterial lysates (soluble fraction) byaffinity chromatography using Ni-NTA columns (Qiagen Cat #30600) as permanufacturer's protocol. N-terminal sequencing indicated that, for oneconstruct (HuIL-1F8A7/FpH), the N-terminal Met was removed (designatedparenthetically in Table 2). Purified polypeptides were tested foractivity in a reporter assay substantially as described in Example 2;results are shown in Table 5 below.

The pH-SUMO construct, encoding HuIL-1F8R5 was introduced into E. coliDH10B. Expression was carried out using Overnight Express™ AutoinductionSystem (Novagen. Darmstadt, Germany, cat#71300-3) per manufacturer'sprotocol. The E. coli cells were centrifuged and frozen. Frozen E. colicell pellets were thawed in TBS made in Roche Complete EDTA freeprotease inhibitor cocktail. After thawing, benzonase was added. Cellswere lysed via passage through a Microfluidics microfluidizer 110Ldevice (MFIC Corporation, Newton, Mass.) and resulting lysates clarifiedvia centrifugation. Supernatants were sterile filtered and loaded onto 5mL HisTrap (GE Biosciences, Piscataway, N.J.) Ni-sepharose columnsequilibrated in Tris, NaCl, imidazole pH 7.4. Columns were washed withimidazole in TBS. Protein was eluted with a imidazole linear gradient.Eluted fractions were pooled and dialyzed into PBS using MWCOSlide-A-Lyzers (Pierce Biotechnology, Inc., Rockland, Ill.). LifeSensorsSUMO Protease-1 was used to cleave the His-SUMO fusion partner fromIL-1F6 variant. Cleavage reaction products were dialyzed into TBScontaining EDTA, pH 7.4 using MWCO Slide-A-Lyzers. Dialyzed pools werethen passed back over HisTrap Ni-Sepharose columns and flow-throughfractions containing pure liberated IL-1F6 variant were retained. andtested for activity as described in Example 2B below. Results are shownin Table 7 below.

Example 1C Preparation of IL-1F9 Variants

Variants of IL-1F9 comprising a C-terminal FLAG-polyHis tag wereprepared in a substantially similar manner as described in Example 1Aabove, using as a template a cDNA clone encoding huIL-1F9 (AF200492).HuIL-1F9S18 (lacking a C-Terminal FLAG-polyHis tag), using as a templatea cDNA clone encoding huIL-1F9 (AF200492), was prepared and purified ina substantially similar manner to HuIL-1F6K6 described in Example 1Aabove except that an extra thymine base pair was removed using a sitedirected mutagenesis kit according to the manufacturer's instructions(Stratagene, LaJolla, Calif., cat#200523). The resulting clone was cutwith restriction enzymes NdeI and Hind III and subcloned back into thepET-SUMO vector (Invitrogen, Carlsbad, Calif.). The various N-terminaldeletion variants are shown in Table 3 below:

TABLE 3 Amino Acid Sequence of IL-1F9 Variants IL-1F9 Variant  Polypeptide:   (Hu indicates human; WT indicates full lengthwild type polypeptide, FpH indicates a FLAG poly His tag., the bold,italicized, upper case N-Terminal amino acid sequence of IL-1F9 Variantsletter and number refers (... indicates intervening aminoto the indicated amino acids of human IL-1F9 (SEQ ID NO 4);acid and its position in ( ) indicate that the enclosed aminoSEQ ID NO 4 where the acid is removed; FLAG indicates theposition is relative to the SEQ amino acid sequence DYKDDDDK; N-Terminal amino acid ID polyHis indicates a chain of multipleat position one of SEQ NO: histidines, typically 6.) ID NO 4.) 33MRGTPGDADGGGRAVYQSMCKPITGT...FELNIND-FLAG-polyHis HuIL-1F9WT/FpH 34              (M)SMCKPITGT...FELNIND-FLAG-polyHis HuIL-1F9

/FpH 35                  MMCKPITGT...FELNIND-FLAG-polyHis HuIL-1F9

/FpH 36                 (M)CKPITGT...FELNIND-FLAG-polyHis HuIL-1F9

/FpH 37                  SMCKPITGT...FELNIND HuIL-1F9

The constructs were expressed in E. coli, and the expressed polypeptidespurified from bacterial lysates (soluble fraction) as describedpreviously. Similar to observations for HuIL-1F8, N-terminal sequencingindicated that, for one construct HuIL-1F9C20/FpH), the N-terminal Metwas removed from the polypeptide. Purified polypeptides were also testedfor activity in a reporter assay substantially as described in Example2. Results are shown in Table 5 below.

The pH-SUMO construct, encoding HuIL-1F9S18 was introduced into E. coliDH10B. Expression was carried out using Overnight Express™ AutoinductionSystem (Novagen, Darmstadt, Germany, cat#71300-3) per manufacturer'sprotocol. The E. Coli cells were centrifuged and frozen. Frozen E. colicell pellets were thawed in TBS made in Roche Complete EDTA freeprotease inhibitor cocktail. After thawing, benzonase was added. Cellswere lysed via passage through a Microfluidics microfluidizer 110Ldevice (MFIC Corporation, Newton, Mass.) and resulting lysates clarifiedvia centrifugation. Supernatants were sterile filtered and loaded onto 5mL HisTrap (GE Biosciences, Piscataway, N.J.) Ni-sepharose columnsequilibrated in Tris, NaCl, imidazole pH 7.4. Columns were washed withimidazole in TBS. Protein was eluted with a imidazole linear gradient.Eluted fractions were pooled and dialyzed into PBS using MWCOSlide-A-Lyzers (Pierce Biotechnology, Inc., Rockland, Ill.). LifeSensorsSUMO Protease-1 was used to cleave the His-SUMO fusion partner fromIL-1F6 variant. Cleavage reaction products were dialyzed into TBScontaining EDTA, pH 7.4 using MWCO Slide-A-Lyzers. Dialyzed pools werethen passed back over HisTrap Ni-Sepharose columns and flow-throughfractions containing pure liberated IL-1F6 variant were retained. andtested for activity as described in Example 2B below. Results are shownin Table 7 below.

Example 1D Preparation of IL-1F5 Variants

Variants of IL-1F5 comprising a C-terminal FLAG-polyHis tag wereprepared in a substantially similar manner as described in Example 1Aabove, using as a template a cDNA clone encoding huIL-1F5 (AF201830).HuIL-1F5V2, using as a template a cDNA clone encoding huIL-1F5(AF201830), was prepared and purified in a substantially similar mannerto HuIL-1F6K6 described in Example 1A The various N-terminal deletionvariants are shown in Table 4 below:

TABLE 4 Amino Acid Sequence of IL-1F5 Variants IL-1F5 VariantPolypeptide: (Hu indicates human; WT indicates full length wildtype polypeptide, FpH indicates a FLAG poly Histag; the bold, italicized,N-Terminal amino acid sequence of IL-1F5 Variants upper case letter and(... indicates intervening amino  number refers to theacids of human IL-1F5 (SEQ ID NO indicated amino acid and1);( ) indicated that the enclosed  its position in SEQ ID NO  amino acid is removed; FLAG 1, where the position is SEQindicates the amino acid sequence relative to the N-Terminal IDDYKDDDDK; polyHis indicates a chain of  amino acid at position one NO:multiple histidines, typically 6.) of SEQ ID NO 1.) 38  MVLSGALCFRMKDSA... FYFQQCD-FLAG-polyHis HuIL-1F5

/FpH 39 (M)VLSGALCFRMKDSA... FYFQQCD-FLAG-polyHis HuIL-1F5

/FpH 40    MLSGALCFRMKDSA... FYFQQCD-FLAG-polyHis HuIL-1F5

/FpH 41   (M)SGALCFRMKDSA... FYFQQCD-FLAG-polyHis HuIL-1F5

/FpH 42    (M)GALCFRMKDSA... FYFQQCD-FLAG-polyHis HuIL-1F5

/FpH 43    VLSGALCFRMKDSA... FYFQQCD HuIL-1F5

HuIL-1F5M1/FpH was expressed in E. coli as a Glutathione-S-transferase(hereinafter “GST”) fusion. The GST domain was cleaved off by digestionwith Factor Xa. HuIL-1F5V2/FpH was expressed in COS-1 cells. Theremaining constructs were expressed as described above. A full-lengthform was also expressed in E. coli, and found to have the sameN-terminal sequence as HuIL-1F5V2FpH; this form was referred to asHuIL-1F5WT/FpH. The polypeptides were purified and tested for theirability to inhibit the activation of IL-1Rrp2 by IL-1F8 in a reporterassay substantially as described in Example 2. In this assay, eachpurified variant of IL-1F5 was added to. IL-1Rrp2 transfected Jurkatcells at 5000, 500, or 50 ng/mL, and the cells were pre-incubated for 15minutes. At that time, IL-1F8 (untagged, full-length—expressed in E.coli; HuIL-1F8WT/FpH) was added at a concentration of 150 ng/mL. Thecells were then incubated with the F8/F5 mixtures for five hours at 37°C. Cell lysates were assayed for luciferase activity as previouslyreported (Towne et al. 2004 J Biol Chem 279(14):13677)). Ratios of F5:F8tested were 33.3:1, 3.3:1, and 0.33:1. Results are shown in Table 5below.

The pH-SUMO construct, encoding HuIL-1FV2 was introduced into E. coliDH10B. Expression was carried out using Overnight Express™ AutoinductionSystem (Novagen, Darmstadt, Germany, cat#71300-3) per manufacturer'sprotocol. The E. Coli cells were centrifuged and frozen. Frozen E. colicell pellets were thawed in TBS made in Roche Complete EDTA freeprotease inhibitor cocktail. After thawing, benzonase was added. Cellswere lysed via passage through a Microfluidics microfluidizer 110Ldevice (MFIC Corporation, Newton, Mass.) and resulting lysates clarifiedvia centrifugation. Supernatants were sterile filtered and loaded onto 5mL HisTrap (GE Biosciences, Piscataway, N.J.) Ni-sepharose columnsequilibrated in Tris, NaCl, imidazole pH 7.4. Columns were washed withimidazole in TBS. Protein was eluted with a imidazole linear gradient.Eluted fractions were pooled and dialyzed into PBS using MWCOSlide-A-Lyzers (Pierce Biotechnology, Inc., Rockland, Ill.). LifeSensorsSUMO Protease-1 was used to cleave the His-SUMO fusion partner fromIL-1F6 variant. Cleavage reaction products were dialyzed into TBScontaining EDTA, pH 7.4 using MWCO Slide-A-Lyzers. Dialyzed pools werethen passed back over HisTrap Ni-Sepharose columns and flow-throughfractions containing pure liberated IL-1F6 variant were retained. andtested for activity as described in Example 2B below. Results are shownin Table 7 below.

Example 2 Example 2A Luciferase Assay of IL-1F Variants

This Example describes a reporter assay used to evaluate the activity ofIL-1 family member variants, substantially as described in Towne et al.,J Biol Chem. 279(14):13677 (2004) herein incorporated by reference inits entirety. Briefly, Jurkat E6.1 cells (7×10⁵) are transientlytransfected via FuGENE 6 (Roche Diagnostics, Basel, Switzerland) as permanufacturer's protocol (i.e., cells are transfected with 200 ngreporter plasmid and 400 ng of either IL-1Rrp2-encoding or empty vectorplasmids with a 1:3 DNA/FuGENE 6 ratio). Seventeen hours aftertransfection, cells are stimulated with the indicated cytokines orcytokine variants for five hours. Cells are lysed and luciferaseactivity is assessed using reporter lysis buffer (Promega) andLuciferase Assay Reagent (Promega). The results reported hereinrepresent duplicate samples.

TABLE 5 Variant HuIL-1F SEQ ID Biological start VariantN-Terminal Sequence NO: Activity* Position ** HuIL-1F5~~~~~~~~MVLSGALCFR

KDSALKVLYLHNN 44 none -10

/FpH HuIL-1F5 ~~~~~~~~~VLSGALCFR

KDSALKVLYLHNN 45 >FL  -9

/FpH HuIL-1F5 ~~~~~~~~~MLSGALCFR

KDSALKVLYLHNN 46 >FL  -9

/FpH HuIL-1F5 ~~~~~~~~~~~SGALCFR

KDSALKVLYLHNN 47 none  -7

/FpH HuIL-1F5 ~~~~~~~~~~~~GALCFR

KDSALKVLYLHNN 48 none  -6

/FpH HuIL-1F6 ~~~~~~~~MKIDTPQRGS

QDINHRVWVLQDQ 49 =FL -10

/FpH HuIL-1F6 ~~~~~~~~~MIDTPQRGS

QDINHRVWVLQDQ 50 >>FL  -9

/FpH HuIL-1F6 ~~~~~~~~~~MDTPQRGS

QDINHRVWVLQDQ 51 =FL  -8

/FpH HuIL-1F8 ~~~~~~~MQREAAPKSYA

RDSRQMVWVLSGN 52 =FL -11

/FpH HuIL-1F8 ~~~~~~~~MREAAPKSYA

RDSRQMVWVLSGN 53 =FL -10

/FpH HuIL-1F8 ~~~~~~~~~MEAAPKSYA

RDSRQMVWVLSGN 54 >>FL  -9

/FpH HuIL-1F8 ~~~~~~~~~~~AAPKSYA

RDSRQMVWVLSGN 55 <FL  -7

/FpH HuIL-1F9 ~~~~~~~~~SMCKPITGT

NDLNQQVWTLQGQ 56 >>FL  -9

/FpH HuIL-1F9 ~~~~~~~~~MMCKPITGT

NDLNQQVWTLQGQ 57 >>FL  -9

/FpH HuIL-1F9 ~~~~~~~~~~~CKPITGT

NDLNQQVWTLQGQ 58 <FL  -7

/FpH *Biological activity is expressed relative to the activity of therelevant full-length polypeptide (FL). ** Indicates the position of thevariant N-terminal amino acid relative to the Methionine or Isoleucineof the consensus sequence @XD. The aliphatic residue (met or ile) isindicated by enclosure in a box.

Analysis of these results indicated that all variants with enhancedactivity relative to the relevant full length IL-1 family member have anN-terminal sequence that begins at position −9 relative to the aliphaticamino acid of the @XD consensus sequence (the aliphatic amino acid isshown as an ‘@’ in the consensus sequence of FIG. 1 and enclosed in abox in the sequences of Table 5 above). There were sufficient datapoints available for some of the constructs to allow calculation of EC₅₀values; for the remaining constructs, the data were insufficient forsuch calculations. The relevant constructs and their EC₅₀s are shown inTable 6 below.

TABLE 6 EC₅₀ EC₅₀ EC₅₀ Assayed (ng/ Assayed (ng/ Assayed (ng/ IL-1Polypeptide: ml) Polypeptide: ml) Variants: ml) F6 HuF6WT >212HuF6WT/ >136 HuF6I7/ 2.8 (untagged): FpH FpH F8 HuF8WT >193 HuF8WT/ >258HuF8I7/ 0.2 (untagged): FpH FpH F9 HuF9WT >544 HuF9WT/ >332 HuF9S718/1.0 (untagged): FpH FpH HuF9M19/ 1.1 FpH

These results demonstrated that the length of the N-terminus isimportant for biological activity of both agonistic and antagonisticIL-1 family members that signal via IL-1Rrp2.

Example 2B IL-8 ELISA Assay of IL-1F Variants

A stable cell line expressing human IL-1Rrp2 off an inducible promoterwas generated in Jurkat T-REx™ cells (Invitrogen). T-RExTm cell linesstably express the tetracycline repressor protein and, therefore, allowfor inducible expression of the gene of interest (IL-1Rrp2) withdoxycycline.

In order to access the activity of the IL-1F ligands IL-1Rrp2 expressingJurkat T-RExTm cells were induced with doxycycline 24 hrs. prior to use.Cells were seeded at 200,000 cells/well in a 96-well round bottom tissueculture plate. The IL-1F ligands (full length (WT) untagged, full length(WT) tagged, truncated tagged or SUMO generated truncated untaggedprotein) were added to the wells as follows: full length IL-1F6, F8 andF9 were added at 50 μg/mL with an 8 point dilution series at 1:5dilutions and truncated ligands were started at 2 μg/mL with 1:5dilutions for 8 points. The cells were incubated with the ligands at 37°C., 5% CO2 for 24 hrs. Following incubation, cells were spun down andthe supernatants were collected for analysis using QuantiGlo Human IL-8ELISA Kit (R&D Systems) per the manfacture's instructions The resultsfor the tested polypeptides and variants are given as indicated in Table7 below.

TABLE 7 IL-8 Elisa Assay Results N-terminal polypeptide sequenceA box frames the aliphatic amino  acid which is indicated as  SEQ @of the consensus sequence: ID Polypeptide @XD where @ may be M or 1,EC₅₀ NO: Assayed: and X is one amino acid. (μg/mL) 59 HuIL-1F6 WT            MEKALKIDTPQQGS

QDINHRVWVLQDQ 12.94 50 HuIL-1F6             ~~~~~MIDTPQRGS

QDINHRVWVLQDQ 0.060

/FpH 60 HuIL-1F6K6             ~~~~~KIDTPQQGS

QDINHRVWVLQDQ 0.000164 61 HuIL-1F8WT              MNPQREAAPKSYA

RDSRQMVWVLSGN 10.47 54 HuIL-1F8             ~~~~~MEAAPKSYA

RDSRQMVWVLSGN 0.002

/FpH 62 HuIL-1F8R5             ~~~~~REAAPKSYA

RDSRQMVWVLSGN 0.000129 63 HuIL-1F9WT MRGTPGDADGGGRAVYQSMCKPITGT

NDLNQQVWTLQGQ 4.072 56 HuIL-1F9 ~~~~~~~~~~~~~~~~~SMCKPITGT

NDLNQQVWTLQGQ 0.017

/FpH 56 HuIL-1F9

~~~~~~~~~~~~~~~~~SMCKPITGT

NDLNQQVWTLQGQ 0.001

These results demonstrated that the length of the N-terminus isimportant for biological activity of both agonistic and antagonisticIL-1 family members that signal via IL-1Rrp2 and that the particularN-terminal amino acid is not important. Further, these resultsdemonstrate that polypeptides that do not comprise a C-terminal tag aremore active than corresponding polypeptides comprising a C-terminal tag.

Example 3 Preparation of Monoclonal Antibodies

IL-1 family member polypeptides may be employed as immunogens ingenerating monoclonal antibodies by conventional techniques, e.g.,techniques described in U.S. Pat. No. 5,599,905, hereby incorporated byreference. It is recognized that polypeptides in various forms may beemployed as immunogens, e.g., full length proteins, fragments thereof,fusion proteins thereof such as Fc fusions, cells expressing therecombinant protein on the cell surface, etc. Examples of usefulpeptides include those shown in FIG. 1.

To summarize an example of such a procedure, an N-terminal peptide of anIL-1Rrp2 requiring IL-1 family member, optionally having an additionalC-terminal cysteine residue to facilitate conjugation, is conjugated tomaleiimide-activated keyhole limpet hemocyanin (KLH; obtainable forexample from Pierce Biotechnology Inc., Rockford, Ill.) to yield animmunogen. For a first immunization, 100 micrograms of immunogen(containing 50 micrograms of peptide) is emulsified in complete Freund'sadjuvant (CFA) at 1:1 ratio by volume and injected subcutaneously in afinal volume of 200 microliters for each mouse.

Immunized animals are boosted three to four more times with additionalimmunogen to increase the antigen-specific response, at intervals of twoto four weeks (although longer intervals may be employed. For example, asecond injection of 50 micrograms of immunogen (containing 25 microgramsof peptide) mixed with incomplete Freund's adjuvant in a final volume of200 ul is injected subcutaneously into each mouse about four weeks afterthe primary immunization. A third injection (20 micrograms of immunogencontaining 10 micrograms of peptide mixed with an adjuvant such as Ribiadjuvant) may be given by subcutaneous and/or intraperitoneal route fromabout 10 to 30 days after the second injection. If desired, a fourthinjection (20 micrograms of immunogen containing 10 micrograms ofpeptide mixed with incomplete Freund's adjuvant) may be given bysubcutaneous and/or intraperitoneal route from about 14 to about 28 daysafter the third injection. A final injection is given, usually aboutfive days prior to fusion, utilizing 50 micrograms of immunogencontaining 25 micrograms of peptide in PBS, by intraperitonealinjection.

Serum samples may be periodically taken by retro-orbital bleeding ortail-tip excision for testing by peptide ELISA (enzyme-linkedimmunosorbent assay), or another suitable assay, to evaluate antibodytiter. At the time of fusion, the animals are sacrificed, splenocytesharvested, and fused to the murine myeloma cell line SP2/O (ATCC CRL1581). The resulting hybridoma cell lines are plated in multiplemicrotiter plates in a HAT selective medium (hypoxanthine, aminopterin,and thymidine) to facilitate proliferation of spleen cell-myeloma hybridcells.

Hybridoma clones thus generated are screened for reactivity with theN-terminal portion of the relevant IL-1 family member. Initial screeningof hybridoma supernatants may utilize a peptide ELISA, a whole cellELISA and/or a cell-based assay suitable for high-throughput screening(fluorometric microvolume assay technology or FMAT, substantially asdescribed by Fiscella, et al., Nature Biotechnology 21:302-307 (2003).Hybridomas that are positive in this screening method may be furthercultured to provide larger amounts of antibody, which can then bepurified as described below and screened by additional cell-basedassay(s) (for example, a reporter assay, or another assay for biologicalactivity of an IL-1 family member).

Selected hybridomas can be further cloned and tested to ensure stableproduction monoclonal antibody. Hybridomas can be cultured in vitro, orpass aged as ascites fluid in suitable host mammals. The resultingmonoclonal antibodies may be purified by ammonium sulfate precipitationfollowed by gel exclusion chromatography, and/or affinity chromatographybased on binding of antibody to Protein G, for example.

Example 4 Purification of Hybridoma Antibodies for Screening

Hybridoma cells are cultured for a time and under conditions to yield asample of about 35 ml of hybridoma supernatant fluid. To each sample isadded 12 ml of 4×-Protein A Binding Buffer (1.6 M citric acid, 100 mMtris, pH 9.15) and about 300 μl of a 67% slurry of MabSelect™ Media (GEHealthcare, Piscataway, N.J.). The resulting slurry is rotated gentlyover night at 4° C.

After overnight incubation, the samples are centrifuged to sediment theresin and the monoclonal antibodies bound thereto, for example at 2,000RPM in a G3.8 centrifuge rotor (Beckman Coulter, Fullerton, Calif.) for5 minutes at 4° C. with no brake. All but about 300 μl of thesupernatant fluid is removed and the resin is resuspended to form aconcentrated slurry.

The concentrated slurry is transferred to a microcentrifuge tube andsufficient 1×-Protein A Binding Buffer (400 mM citric acid, 25 mM tris,pH 8.9) is added to bring the total volume up to about 1 ml. The slurryis resuspended, then centrifuged at about 14,000 g for 5 seconds. Thesupernatant fluid is removed from the resulting pellet, which is washeda total of three times in a similar manner (i.e. by resuspending inabout 1 ml of 1×-Protein A Binding Buffer, centrifuging, removingsupernatant and resuspending in fresh buffer).

After three washes, the pellet is resuspended in 400 μl Elution Buffer(200 mM formic acid) and agitated for 10 min at room temperature, thencentrifuged at 14,000 g for 5 seconds. The supernatant is carefullyremoved as eluate, and the pellet is eluted again in a manner similar tothat described above for a total of three elution cycles. The eluatesfrom the three elution cycles are combined, centrifuge at 14,000 g for 5min room temperature and transferred to a fresh tube. The pH is adjustedto 7.8-8.2 by adding 2 M tris base (235 mM_(f)) and mixing quickly. Thesamples are again centrifuged at 14,000 g for 5 min at room temperature,and designated as pH Shift Soluble. A spectral scan of each sample(diluted by adding 20 μl of the sample to 700 μl water) is run from 250to 350 nm, and protein concentration is verified by loading 0.5 μg eachantibody-containing sample on a reducing 4-20% SDS-PAGE gel with anappropriate antibody standards.

Each reference cited herein is incorporated by reference in its entiretyfor all that it teaches and for all purposes.

What is claimed is:
 1. An isolated IL-1F5 polypeptide that antagonizessignal transduction/activation through IL-1Rrp2, the IL-1F5 polypeptidecomprising an amino acid sequence having a methionine at position ten ofthe amino acid sequence, position ten being relative to the N-terminalamino acid at position one of the amino acid sequence.
 2. The isolatedIL-1F5 polypeptide of claim 1, wherein the N-terminal amino acid atposition one is valine.
 3. The isolated IL-1F5 polypeptide of claim 1,wherein the N-terminal amino acid at position one is a methionine. 4.The isolated IL-1F5 polypeptide of claim 1, wherein the amino acid atposition two relative to the N-terminal amino acid at position one, is aleucine.
 5. The isolated IL-1F5 polypeptide of claim 1, wherein theisolated IL-1F5 polypeptide antagonizes signal transduction/activationthrough IL-1Rrp2 more than an IL-1F5 polypeptide consisting of SEQ IDNO
 1. 6. The isolated IL-1F5 polypeptide of claim 1 comprising an aminoacid sequence selected from the group consisting of: SEQ ID NO 6, SEQ IDNO 7, SEQ ID NO 8 and SEQ ID NO
 9. 7. A nucleic acid sequence encodingthe IL-1F5 polypeptide of claim
 1. 8. A recombinant vector that directsexpression of the nucleic acid of claim
 7. 9. A host cell transfected ortransduced with the vector of claim
 8. 10. A method of producing anIL-1F5 polypeptide comprising culturing the host cell of claim 9 underconditions promoting expression and isolating the expressed IL-1F5polypeptide.
 11. A composition comprising the polypeptide of claim 1 anda physiologically acceptable diluent, excipient or carrier.
 12. A methodof treating an inflammatory or autoimmune condition in a subject whereinthe inflammatory or autoimmune condition is mediated by IL-1Rrp2,comprising administering to the subject in an amount of the IL-1F5polypeptide of claim 1 sufficient to reduce at least one symptom of theinflammatory or autoimmune condition in the subject.
 13. The method ofclaim 12, wherein the condition to be treated is an inflammatorycondition of the skin, lungs or airways mediated by IL-1Rrp2.
 14. Themethod of claim 12, wherein the condition to be treated is selected fromthe group consisting of psoriasis, seborrheic dermatitis, atopicdermatitis, including chronic atopic dermatitis, allergic contactdermatitis, lichen simplex chronicus, pityriasis rubra pilaris, nummulardermatitis, asthma, allergic rhinitis, gastro-esophageal reflux disease,arthritic conditions including, rheumatoid arthritis, psoriaticarthritis, and osteoarthritis.
 15. The method of claim 12, wherein thesubject is human.
 16. An isolated IL-1F6 polypeptide that agonizessignal transduction/activation through IL-1Rrp2, wherein the IL-1F6polypeptide comprises an amino acid sequence having a isoleucine atposition ten of the amino acid sequence, position ten being relative tothe N-terminal amino acid at position one of the amino acid sequence.17. The isolated IL-1F6 polypeptide of claim 16, wherein the N-terminalamino acid at position one is a lysine.
 18. The isolated IL-1F6polypeptide of claim 16, wherein the N-terminal amino acid at positionone is a methionine.
 19. The isolated IL-1F6 polypeptide of claim 16,wherein the amino acid at position two relative to the N-terminal aminoacid at position one of the amino acid sequence, is an isoleucine. 20.The isolated IL-1F6 polypeptide of claim 16, wherein the isolated IL-1F6polypeptide agonizes signal transduction/activation through IL-1Rrp2more than an IL-1F6 polypeptide consisting of SEQ ID NO
 2. 21. Theisolated IL-1F6 polypeptide of claim 16, comprising an amino acidsequence selected from the group consisting of: SEQ ID NO 10, SEQ ID NO11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67and SEQ ID NO
 68. 22. A nucleic acid sequence encoding the IL-1F6 ofclaim
 16. 23. A recombinant vector that directs expression of thenucleic acid of claim
 22. 24. A host cell transfected or transduced withthe vector of claim
 23. 25. A method of producing an IL-1F6 polypeptidecomprising culturing the host cell of claim 24 under conditionspromoting expression and isolating the expressed IL-1F6 polypeptide. 26.A composition comprising the polypeptide of claim 16 and aphysiologically acceptable diluent, excipient or carrier.
 27. A methodof stimulating the immune system of an immunosuppresed patient,comprising administering the IL-1F6 polypeptide according to claim 16 toan immunosuppresed patient in an amount sufficient to stimulate thepatient's immune system.
 28. An antibody that specifically binds anIL-1F6 polypeptide and prevents proteolytic cleavage thereof.
 29. Theantibody of claim 28, wherein the antibody is a monoclonal antibody. 30.A composition comprising the antibody of claim 28 and a physiologicallyacceptable diluent, excipient or carrier.
 31. A method of treating aninflammatory or autoimmune condition mediated by IL-1Rrp2, comprisingadministering the IL-1F6 antibody of claim 29 to a subject in an amountsufficient to ameliorate at least one symptom of the condition.
 32. Themethod of claim 31, wherein the condition to be treated is aninflammatory condition of the skin, lungs or airways mediated byIL-1Rrp2.
 33. The method of claim 31, wherein the condition to betreated is selected from the group consisting of psoriasis, seborrheicdermatitis, atopic dermatitis, including chronic atopic dermatitis,allergic contact dermatitis, lichen simplex chronicus, pityriasis rubrapilaris, nummular dermatitis, asthma, allergic rhinitis,gastro-esophageal reflux disease, arthritic conditions including,rheumatoid arthritis, psoriatic arthritis, and osteoarthritis.
 34. Themethod of claim 31, wherein the subject is human.
 35. An isolated IL-1F8polypeptide that agonizes signal transduction/activation throughIL-1Rrp2, wherein the IL-1F8 polypeptide comprises an amino acidsequence having a isoleucine at position ten of the amino acid sequence,position ten being relative to the N-terminal amino acid at position oneof the amino acid sequence.
 36. The isolated IL-1F8 polypeptide of claim35, wherein the N-terminal amino acid at position one is an arginine.37. The isolated IL-1F8 polypeptide of claim 35, wherein the N-terminalamino acid at position one is a methionine.
 38. The isolated IL-1F8polypeptide of claim 35, wherein the amino acid at position two,position two being relative to the N-terminal amino acid at positionone, is a glutamic acid.
 39. The isolated IL-1F8 polypeptide of claim35, wherein the isolated IL-1F6 polypeptide agonizes signaltransduction/activation through IL-1Rrp2 more than an IL-1F8 polypeptideconsisting of SEQ ID NO
 3. 40. The isolated IL-1F8 polypeptide of claim35, comprising an amino acid sequence selected from the group consistingof: SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO
 17. 41. Anucleic acid sequence encoding the IL-1F8 polypeptide of claim
 35. 42. Arecombinant vector that directs expression of the nucleic acid of claim41.
 43. A host cell transfected or transduced with the vector of claim42.
 44. A method of producing an IL-1F8 polypeptide comprising culturingthe host cell of claim 43 under conditions promoting expression andisolating the expressed IL-1F8 polypeptide.
 45. A composition comprisingthe polypeptide of claim 35 and a physiologically acceptable diluent,excipient or carrier.
 46. A method of stimulating the immune system ofan immunosuppresed patient, comprising administering the IL-1F8polypeptide according to claim 35 to an immunosuppresed patient in anamount sufficient to stimulate the patient's immune system.
 47. Anantibody that specifically binds an IL-1F8 polypeptide and preventsproteolytic cleavage thereof.
 48. The antibody of claim 47, wherein theantibody is a monoclonal antibody.
 49. A composition comprising theantibody of claim 47 and a physiologically acceptable diluent, excipientor carrier.
 50. A method of treating an inflammatory or autoimmunecondition mediated by IL-1Rrp2, comprising administering the IL-1F8antibody of claim 47 to a subject in and amount sufficient to ameliorateat least one symptom.
 51. The method of claim 50, wherein the conditionto be treated is an inflammatory condition of the skin, lungs or airwaysmediated by IL-1Rrp2.
 52. The method of claim 50, wherein the conditionto be treated is selected from the group consisting of psoriasis,seborrheic dermatitis, atopic dermatitis, including chronic atopicdermatitis, allergic contact dermatitis, lichen simplex chronicus,pityriasis rubra pilaris, nummular dermatitis, asthma, allergicrhinitis, gastro-esophageal reflux disease, arthritic conditionsincluding, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis.
 53. An isolated IL-1F9 polypeptide that agonizes signaltransduction/activation through IL-1Rrp2, wherein the IL-1F9 polypeptidecomprises an amino acid sequence having a isoleucine at position ten ofthe amino acid sequence, position ten being relative to the N-terminalamino acid at position one of the amino acid sequence.
 54. The isolatedIL-1F9 polypeptide of claim 53, wherein the N-terminal amino acid atposition one is a serine.
 55. The isolated IL-1F9 polypeptide of claim53, wherein the N-terminal amino acid at position one is a methionine.56. The isolated IL-1F9 polypeptide of claim 53, wherein the amino acidat position two relative to the N terminal amino acid at position one,is a methionine.
 57. The isolated IL-1F9 polypeptide of claim 53,wherein the isolated IL-1F9 polypeptide agonizes signaltransduction/activation through IL-1Rrp2 more than an IL-1F9 polypeptidehaving an amino acid sequence consisting of SEQ ID NO
 4. 58. Theisolated IL-1F9 polypeptide of claim 53, comprising an amino acidsequence selected from the group consisting of: SEQ ID NO 18, SEQ ID NO19, SEQ ID NO 20 and SEQ ID NO
 21. 59. A nucleic acid sequence encodingthe IL-1F9 polypeptide of claim
 53. 60. A recombinant vector thatdirects expression of the nucleic acid of claim
 59. 61. A host celltransfected or transduced with the vector of claim
 60. 62. A method ofproducing an IL-1F9 polypeptide comprising culturing the host cell ofclaim 52 under conditions promoting expression and isolating theexpressed IL-1F9 polypeptide.
 63. A composition comprising thepolypeptide of claim 53 and a physiologically acceptable diluent,excipient or carrier.
 64. A method of stimulating the immune system ofan immunosuppresed patient, comprising administering the IL-1F9polypeptide according to claim 53 to an immunosuppresed patient in anamount sufficient to stimulate the patient's immune system.
 65. Anantibody that specifically binds an IL-1F9 polypeptide and preventsproteolytic cleavage thereof.
 66. The antibody of claim 65, wherein theantibody is a monoclonal antibody.
 67. A composition comprising theantibody of claim 65 and a physiologically acceptable diluent, excipientor carrier.
 68. A method of treating an inflammatory or autoimmunecondition mediated by IL-1Rrp2, comprising administering the IL-1F9antibody of claim 65 to a subject in an amount sufficient to ameliorateat least one symptom of the condition.
 69. The method of claim 68,wherein the condition to be treated is an inflammatory condition of theskin, lungs or airways mediated by IL-1Rrp2.
 70. The method of claim 68,wherein the condition to be treated is selected from the groupconsisting of psoriasis, seborrheic dermatitis, atopic dermatitis,including chronic atopic dermatitis, allergic contact dermatitis, lichensimplex chronicus, pityriasis rubra pilaris, nummular dermatitis,asthma, allergic rhinitis, gastro-esophageal reflux disease, arthriticconditions including, rheumatoid arthritis, psoriatic arthritis, andosteoarthritis.
 71. The method of claim 68, wherein the subject ishuman.
 72. A method of identifying a protease that cleaves an IL-1family member comprising contacting a source of the protease with theIL-1 family member under conditions promoting proteolytic cleavage ofthe IL-1 family member, and determining if the IL-1 family member hasbeen proteolytically cleaved.
 73. A method of identifying an inhibitorof a protease that cleaves an IL-1 family member comprising contactingthe protease with the IL-1 family member in the presence, and absence,of a molecule that is a potential inhibitor, under conditions promotingproteolytic cleavage of the IL-1 family member, and determining if theIL-1 family member has been proteolytically cleaved, wherein if the IL-1family member is not cleaved or is cleaved to a lesser degree in thepresence of the molecule, the molecule is an inhibitor.